Modification of enzymatic crosslinkers for controlling properties of crosslinked matrices

ABSTRACT

Improved matrix or hydrogel that is formed by enzymatic crosslinking of polymers wherein the crosslinking enzyme molecules have been modified for the purpose of improving the crosslinking density, mechanical properties, or other properties of the matrix, and/or to provide improved control over the rate and/or extent of crosslinking, wherein the enzyme molecules are modified to alter the perceived volume of the enzyme molecules in the crosslinked matrix being formed. Methods of production and of use are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application is a national phase of, and claims priority from, PCTApplication No. PCT/IB2010/056008, filed on Dec. 22 2010, which claimspriority from U.S. Provisional Application No. 61/289,368, filed on Dec.22 2009, and both of which are hereby incorporated by reference as iffully set forth herein.

BACKGROUND

Utility of Enzyme Crosslinked Matrices

Enzyme crosslinked matrices are formed in a variety of applications inthe food, cosmetic, and medical industries. In medical applications inparticular, enzyme crosslinked hydrogels are widely used in a variety ofmedical applications including tissue sealants and adhesives,haemostatic preparations, matrices for tissue engineering or platformsfor drug delivery. While some hydrogels such as gelatin and poloxamermay be formed as a result of physical interactions between the polymerchains under specific conditions, e.g change in temperature, mostpolymer solutions must be crosslinked in order to form hydrogels. Inaddition to the actual formation of the solid gel, implantable hydrogelsmust be resistant to the conditions that are prevalent in the tissuewhere they are applied, such as mechanical stress, temperature increase,and enzymatic and chemical degradation. For this reason, in many casesit is necessary to crosslink the hydrogel matrices. The crosslinking maybe done outside the body by pre-casting or molding of hydrogels. Thisapplication is used mainly for tissue engineering or drug deliveryapplications. Alternatively, crosslinking may be done inside the body(in situ gelation or crosslinking) where a liquid solution is injectedor applied to the desired site and is cross linked to form a gel.

Gel formation can be initiated by a variety of crosslinking approaches.Chemical approaches to gel formation include the initiation ofpolymerization either by contact, as in cyanoacrylates, or externalstimuli such as photo-initiation. Also, gel formation can be achieved bychemically crosslinking pre-formed polymers using either low molecularweight crosslinkers such as glutaraldehyde or carbodiimide (Otani Y,Tabata Y, Ikada Y. Ann Thorac Surg 1999, 67, 922-6. Sung H W, Huang D M,Chang W H, Huang R N, Hsu J C. J Biomed Mater Res 1999, 46, 520-30.Otani, Y.; Tabata, Y.; Ikada, Y. Biomaterials 1998, 19, 2167-73. Lim, D.W.; Nettles, D. L.; Setton, L. A.; Chilkoti, A. Biomacromolecules 2008,9, 222-30.), or activated substituents on the polymer (Iwata, H.;Matsuda, S.; Mitsuhashi, K.; Itoh, E.; Ikada, Y. Biomaterials 1998, 19,1869-76).

However, chemical crosslinking can be problematic in food, cosmetic, ormedical applications because the cross-linkers are often toxic,carcinogenic, or irritants. Furthermore, they are small molecules thatcan readily diffuse out of the crosslinked matrix and might cause localor systemic damage.

An alternative to chemical crosslinking is the enzymatic crosslinkingapproach. These approaches to initiate gel formation have beeninvestigated based on a variety of different crosslinking enzymes.Examples include enzymatic crosslinking of adhesives, such as musselglue (Strausberg RL, Link RP. Trends Biotechnol 1990, 8, 53-7), or theenzymatic crosslinking of blood coagulation, as in fibrin sealants(Jackson MR. Am J Surg 2001, 182, 1S-7S. Spotnitz W D. Am J Surg 2001,182, 8S-14S Buchta C, Hedrich H C, Macher M, Hocker P, Redl H.Biomaterials 2005, 26, 6233-41.27-30).

Cross-linking of a mussel glue was initiated by the enzymatic conversionof phenolic (i.e., dopa) residues of the adhesive protein into reactivequinone residues that can undergo subsequent inter-protein crosslinkingreactions (Burzio L A, Waite J H. Biochemistry 2000, 39, 11147-53.McDowell L M, Burzio L A, Waite J H, Schaefer J J. Biol Chem 1999,274,20293-5). The enzymes which have been employed in this class ofsealants are tyrosinase on one hand and laccase and peroxidase on theother hand which acts by forming quinones and free radicals,respectively from tyrosine and other phenolic compounds. These in turncan crosslink to free amines on proteins or to similarly modifiedphenolic groups on proteins and polysaccharides.

A second cross-linking operation that has served as a technologicalmodel is the transglutaminase-catalyzed reactions that occur duringblood coagulation (Ehrbar M, Rizzi S C, Hlushchuk R, Djonov V, Zisch AH, Hubbell J A, Weber F E, Lutolf M P. Biomaterials 2007, 28, 3856-66).Biomimetic approaches for in situ gel formation have investigated theuse of Factor XIIIa or other tissue transglutaminases (Sperinde J,Griffith L. Macromolecules 2000, 33, 5476-5480. Sanborn T J, MessersmithP B, Barron A E. Biomaterials 2002, 23, 2703-10).

An additional in situ crosslinked gel formation of particular interestis the crosslinking of gelatin by a calcium independent microbialtransglutaminase (mTG). mTG catalyzes an analogous crosslinking reactionas Factor XIIIa but the microbial enzyme requires neither thrombin norcalcium for activity. Initial studies with mTG were targeted toapplications in the food industry (Babin H, Dickinson E. FoodHydrocolloids 2001, 15, 271-276. Motoki M, Seguro K. Trends in FoodScience & Technology 1998, 9, 204-210.), while later studies consideredpotential medical applications. Previous in vitro studies have shownthat mTG can crosslink gelatin to form a gel within minutes, thegelatin-mTG adhesive can bond with moist or wet tissue, and the adhesivestrength is comparable to, or better than, fibrin-based sealants (Chen TH, Payne G F, et al. Biomaterials 2003, 24, 2831-2841. McDermott M K,Payne G F, et al. Biomacromolecules 2004, 5, 1270-1279. Chen T, Payne GF, et al. J Biomed Mater Res B Appl Biomater 2006, 77, 416-22.). The useof gelatin and mTG as a medical adhesive is described in PCTWO/2008/076407.

One of the disadvantages of using enzymes as the cross-linkers incrosslinked matrix formation is that they may continue the crosslinkingreaction after the desired gel state has been formed. This is often notdesired because excessive crosslinking may result in a stiffer, morebrittle, and less flexible gel. In addition, the mechanical propertiesof the crosslinked matrix will continue to change during the lifetime ofthe gel, making consistent properties difficult to achieve. Thecontinued enzymatic crosslinking beyond the desired crosslinking densityresults from the ability of the enzyme to continue to catalyze thecrosslinking reaction even once a crosslinked matrix or hydrogel hasbeen formed. This depends on the ability of the enzyme to continue todiffuse throughout the matrix even as solution viscosity increasesgreatly. This view is consistent with Hu et al (Hu B H, Messersmith PB.J. Am. Chem. Soc., 2003, 125 (47), pp 14298-14299) who suggested, basedon work done with peptide-grafted synthetic polymer solutions, thatduring incipient network formation resulting from partial cross-linkingof a polymer solution, the solution viscosity rapidly increases whilethe mobility of the transglutaminase rapidly decreases.

The problem of excessive enzymatic crosslinking leading to a reductionin mechanical properties has been previously documented on severaloccasions:

Bauer et al. demonstrated that high levels of microbial transglutaminase(mTG) caused excessive cross-linking of wheat gluten proteins leading toa loss of elasticity and mechanical damage of the gluten networks.(Bauer N, Koehler P, Wieser H, and Schieberle P. Studies on Effects ofMicrobial Transglutaminase on Gluten Proteins of Wheat II RheologicalProperties. Cereal Chem. 80(6):787-790).

Sakai et al. found that a larger quantity of covalent cross-linkingbetween phenols was effective for enhancement of the mechanicalstability, however, further cross-linking between the phenols resultedin the formation of a brittle gel. (Sakai S, Kawakami K. Synthesis andcharacterization of both ionically and enzymatically crosslinkablealginate, Acta Biomater 3 (2007), pp. 495-501)

In the case of cofactor-dependent crosslinking enzymes, such ascalcium-dependent transglutaminase, removing the cofactor, by binding orotherwise, after a certain reaction time can limit the degree ofcrosslinking. However, cofactor removal is frequently not technicallyfeasible in hydrogel formation where the hydrogel may trap the cofactor.When using cofactor-independent enzymes, such as transglutaminasesavailable from microbial origin, limited degrees of crosslinking can beobtained by heat treatment of the reaction system. However, such atreatment induces negative side effects on protein functionality and istherefore undesirable to apply. In addition, not all reaction systemsare suitable to undergo heat treatment.

Other than resulting in excessive crosslinking within the crosslinkedmatrix, continued diffusion of the crosslinked enzyme in the matrixafter the desired crosslinked state has been achieved also can result ina high rate of enzyme diffusion out of the gel, also known as enzymeelution. This can also be problematic as high levels of crosslinkingenzyme released into the body can interact with body tissues and causelocal or systemic damage.

SUMMARY OF INVENTION

There is a need for, and it would be useful to have, an improved enzymecrosslinked composition which could be used for a wide variety ofapplications.

Therefore, there is a need for, and it would be useful to have amechanism to stop enzymatic crosslinking of crosslinked matricesfollowing the initial formation of the solid matrix at a point where thedesired mechanical properties have obtained; and/or to reduce the extentand rate of elution of the enzyme from the solid crosslinked matrix.

The present invention, in at least some embodiments, overcomes the abovedescribed drawbacks of the background art, and provides a solution tothe above technical problems (among its many advantages and withoutwishing to provide a closed list), by providing a matrix or hydrogelthat is formed by enzymatic crosslinking of polymers wherein thecrosslinking enzyme molecules have been modified for the purpose ofimproving the crosslinking density, mechanical properties, or otherproperties of the matrix, and/or to provide improved control over therate and/or extent of crosslinking.

An optional method of altering the enzyme molecules is by modifying theperceived volume of the enzyme molecules in the crosslinked matrix beingformed. The modified perceived volume is preferably determined accordingto the extent of crosslinking of the polymers to form the matrix, suchthat decreased extent of crosslinking, as compared with extent ofcrosslinking with unmodified enzyme molecules, indicates increasedperceived volume.

One method of increasing the perceived volume of the enzyme molecules isby increasing the size and/or the hydrodynamic volume of the moleculesby covalent or non-covalent attachment of at least one molecule ormoiety to the enzyme molecules. The inventors have demonstrated that thedegree of enzymatic crosslinking in hydrogels or crosslinked matricescan be regulated by covalent attachment of molecules to the enzyme suchthat the modification of the enzyme molecules result in a lower ultimatelevel of crosslinking. In this manner, the phenomenon of excessivecrosslinking can be prevented.

Another method of increasing the perceived volume is throughmodification of the electrostatic charge of the enzyme molecules suchthat their net charge is of opposite sign to the net charge on thepolymer or co-polymer chains. This can be achieved by changing theisoelectric point (pI) of the enzyme.

In a non-limiting hypothesis, increasing the perceived volume of theenzyme molecules reduces the mobility or diffusion of the molecules inthe crosslinked matrix or hydrogel. This prevents it from continuing itscrosslinking activity beyond the point where the crosslinking isbeneficial to the desired material properties of the hydrogel.

“Perceived volume” or “effective volume” as defined herein refers to theeffective hydrodynamic volume of the crosslinking enzyme inside thecrosslinked matrix. The perceived volume may be increased by covalent ornon-covalent binding of the enzyme to another molecule, carrier,polymer, protein, polysaccharide and others, prior to the crosslinkingreaction or during the crosslinking reaction.

“Diffusion” or “Mobility” as defined herein refers to the randommolecular motion of the crosslinking enzyme or other proteins, insolution, hydrogen, or matrix that result from Brownian motion.

“Diffusion coefficient” as defined herein refers to a term thatquantifies the extent of diffusion for a single type of molecule underspecific conditions. A non-limiting example of a proxy for measuringenzyme diffusion is by measuring the elution of enzyme from a hydrogel.

“Reduced Mobility” as defined herein refers to a slower molecular motionor smaller diffusion coefficient of a protein or enzyme in a solution orinside a hydrogel. “Size” as defined herein refers to the molecularweight or hydrodynamic volume or perceived volume of a molecule.

“Molecular weight”, abbreviated as MW, as used herein refers to theabsolute weight in Daltons or kilodaltons of proteins or polymers. Forexample, the MW of a PEGylated protein (ie—protein to which one or morePEG (polyethylene glycol) molecules have been coupled) is the MW sum ofall of its constituents.

“Hydrodynamic Volume” as defined herein refers to the apparent molecularweight of a protein or enzyme that may usually be measured using sizeexclusion chromatography. The hydrodynamic volume of a constituentrefers to the diameter or volume the constituent assumes when it is inmotion in a liquid form. “Matrix” as defined herein refers to refers toa composition of crosslinked materials. Generally, when thematrix-forming materials are crosslinked, the composition that includesthese materials transitions from a liquid state to a gel state, therebyforming a “gel,” “hydrogel” or a “gelled composition.” The gel can havecertain viscoelastic and rheological properties that provide it withcertain degrees of durability and swellability. These materials areoften polymers. The matrix may contain materials which are notcrosslinked, sometimes referred to as co-polymers.

“Polymer” as used herein refers to a natural, synthetic orsemi-synthetic molecule, containing a repeatable unit.

“Co-polymer” as used herein refers to a constituent of the matrix whichmay or may not participate in the crosslinking reaction and is usuallynot the main constituent of the matrix. A non-limiting example comprisespolysaccharides such as dextran and/or a cellulosic polymer such ascarboxymethyl cellulose. The co-polymer is preferably not covalentlybound to the enzyme or to the matrix material, such as the protein baseof the matrix. “Carrier” as used herein refers to a polymer, a protein,polysaccharide or any other constituent which binds the crosslinkingenzyme covalently or non-covalently, either before or during thecrosslinking reaction.

“Crosslinking Enzyme” as defined herein refers to an enzyme orcombination of enzymes that can either directly (e.g. bytransglutamination) or indirectly (e.g. through quinone or free radicalformation) crosslink substrate groups on polymer strands into a coherentmatrix, such as a hydrogel.

According to at least some embodiments of the present invention, thereis provided a cross-linked matrix, comprising a substrate polymercrosslinked by a modified enzyme molecule, said modified enzyme moleculehaving a modification that alters a perceived volume of the enzymemolecules in the crosslinked matrix as the matrix is being formedthrough cross-linking of said polymer.

Optionally said modified enzyme molecule has a modification thatincreases an actual size of said modified enzyme molecule. Optionallysaid modified enzyme molecule has a modification that increases ahydrodynamic volume of said modified enzyme molecule. Optionally saidmodified enzyme molecule has a modification that modifies anelectrostatic charge of said modified enzyme molecule to be of oppositesign to a net charge of said substrate polymer by changing theisoelectric point (p1) of said modified enzyme in comparison tounmodified enzyme. Optionally said modification is of the ε-amino groupof lysines of the enzyme through a process selected from the groupconsisting of succinylation (with succinic anhydride), acetylation (withacetic anhydride), carbamylation (with cyanate), reductive alkylation(aldehydes) and treatment with maleic anhydride. Optionally saidmodification is of one or more side chains containing carboxylic acidsof the enzyme to decrease the number of negative charges.

Optionally said modification comprises covalent or non-covalentattachment of at least one molecule or moiety to said modified enzymemolecule. Optionally said modification comprises covalent attachment ofa modifying molecule to said modified enzyme molecule. Optionally saidmodified enzyme molecule has a reduced diffusion rate and a reducedcross-linking rate in comparison to non-modified enzyme, but has atleast similar measured enzyme activity in comparison to non-modifiedenzyme.

Optionally reduced cross-linking rate is at least 10% of thenon-modified enzyme cross-linking rate.

Optionally said modifying molecule comprises a carrier or polymer.Optionally said polymer comprises a synthetic polymer, a cellulosicpolymer, a protein or a polysaccharide. Optionally said cellulosicpolymer comprises one or more of carboxymethyl cellulose, hydroxypropylmethylcellulose, hydroxyethyl cellulose, or methyl cellulose. Optionallysaid polysaccharide comprises one or more of dextran, chondroitinsulfate, dermatan sulfate, keratan sulfate, heparin, heparan sulfate,hyaluronic acid or a starch derivative.

Optionally said modifying molecule comprises PEG (polyethylene glycol).Optionally said PEG comprises a PEG derivative. Optionally said PEGderivative comprises activated PEG. Optionally said activated PEGcomprises one or more of methoxy PEG (mPEG), its derivatives, mPEG-NHS,succinimidyl (NHS) esters of mPEG (mPEG-succinate-NHS),mPEG-glutarate-NHS, mPEG-valerate-NHS, mPEG-carbonate-NHS,mPEG-carboxymethyl-NHS, mPEG-propionate-NHS, mPEG-carboxypentyl-NHS),mPEG-nitrophenylcarbonate, mPEG-propylaldehyde, mPEG-Tosylate,mPEG-carbonylimidazole, mPEG-isocyanate, mPEG-epoxide or a combinationthereof. Optionally said activated PEG reacts with amine groups or thiolgroups on said enzyme. Optionally the molar ratio of said activated PEGto lysine residues of said activated enzyme is in a range of from 0.5 to25. Optionally said activated PEG is monofunctional, heterobifunctional,homobifunctional, or multifunctional. Optionally said activated PEG isbranched PEGs or multi-arm PEGs. Optionally said activated PEG has asize ranging from 1000 dalton to 40,000 dalton.

Optionally the matrix further comprises a co-polymer that is notcovalently bound to said enzyme or to said substrate polymer. Optionallysaid co-polymer comprises a polysaccharide or a cellulosic polymer.Optionally said polysaccharide comprises dextran, chondroitin sulfate,dermatan sulfate, keratan sulfate, heparin, heparan sulfate, hyaluronicacid or a starch derivative. Optionally said cellulosic polymercomprises carboxymethyl cellulose, hydroxypropyl methylcellulose,hydroxyethyl cellulose, methyl cellulose.

Optionally said modified enzyme molecule is modified by cross-linkingsaid modified enzyme molecule to a plurality of other enzyme moleculesto form an aggregate of a plurality of cross-linked enzyme molecules.

Optionally a modification or an extent of modification of said modifiedenzyme molecule affects at least one property of the matrix. Optionallysaid at least one property is selected from the group consisting oftensile strength, stiffness, extent of crosslinking of said substratepolymer, viscosity, elasticity, flexibility, strain to break, stress tobreak, Poisson's ratio, swelling capacity and Young's modulus, or acombination thereof.

Optionally an extent of modification of said modified enzyme determinesmobility of said modified enzyme in, or diffusion from, the matrix.Optionally said modification of said modified enzyme reduces diffusioncoefficient of said modified enzyme in a solution of said modifiedenzyme and said protein or in a matrix of said modified enzyme and saidprotein, in comparison to a solution or matrix of non-modified enzymeand said protein. Optionally an extent of modification of said modifiedenzyme determines one or more matrix mechanical properties. Optionallysaid modified enzyme molecule shows a greater differential ofcrosslinking rate in crosslinked polymer than in solution as compared tonon-modified enzyme molecule.

According to at least some embodiments of the present invention, thereis provided a method for controlling formation of a matrix, comprisingmodifying an enzyme molecule with a modification that alters a perceivedvolume of the enzyme molecules in the crosslinked matrix as the matrixis being formed; mixing said modified enzyme molecule with at least onesubstrate polymer that is a substrate of said modified enzyme molecule;and forming the matrix through crosslinking of said at least onesubstrate polymer by said modified enzyme molecule, wherein said formingthe matrix is at least partially controlled by said modification of saidenzyme molecule. Optionally said modification reduces a crosslinkingrate of said modified enzyme molecule as an extent of crosslinking ofsaid at least one substrate polymer increases. Optionally said modifiedenzyme molecule and said at least one substrate polymer are mixed insolution, such that said modification controls extent of crosslinking ofsaid at least one substrate polymer as a viscosity of said solutionincreases. Optionally said modifying comprises PEGylation of the enzymeat a pH in a range from 7 to 9. Optionally pH of the PEGylation reactionis 7.5 -8.5.

According to at least some embodiments for the method and/or matrix,said at least one substrate polymer comprises a substrate polymerselected from the group consisting of a naturally cross-linkablepolymer, a partially denatured polymer that is cross-linkable by saidmodified enzyme and a modified polymer comprising a functional group ora peptide that is cross-linkable by said modified enzyme. Optionallysaid at least one substrate polymer comprises gelatin, collagen, caseinor albumin, or a modified polymer, and wherein said modified enzymemolecule comprises a modified transglutaminase and/or a modifiedoxidative enzyme. Optionally said at least one substrate polymercomprises gelatin selected from the group consisting of gelatin obtainedby partial hydrolysis of animal tissue or collagen obtained from animaltissue, wherein said animal tissue is selected from the group consistingof animal skin, connective tissue, antlers, horns, bones, fish scales,and a recombinant gelatin produced using bacterial, yeast, animal,insect, or plant systems or any type of cell culture, or any combinationthereof. Optionally said gelatin is of mammalian or fish origin.Optionally said gelatin is of type A (Acid Treated) or of type B(Alkaline Treated). Optionally said gelatin is of 250-300 bloom.Optionally said gelatin has an average molecular weight of 75-150 kda.

Optionally said modified transglutaminase comprises modified microbialtransglutaminase. Optionally said modified polymer is modified to permitcrosslinking by said modified microbial transglutaminase. Optionallysaid modified oxidative enzyme comprises one or more of tyrosinase,laccase, or peroxidase. Optionally said matrix further comprises acarbohydrate comprising a phenolic acid for being cross-linked by saidmodified oxidative enzyme as said at least one substrate polymer.Optionally said carbohydrate comprises one or more of arabinoxylan orpectin. Optionally said enzyme molecule is modified through PEGylationand wherein said PEGylation provides immunogenic masking by masking saidenzyme molecule from an immune system of a host animal receiving thematrix. Optionally said host animal is human.

According to at least some embodiments, there is provided a method forsealing a tissue against leakage of a body fluid, comprising applying amatrix as described herein to the tissue. Optionally said body fluidcomprises blood, such that said matrix is a hemostatic agent.

According to at least some embodiments, there is provided a hemostaticagent or surgical sealant, comprising a matrix as described herein.

According to at least some embodiments, there is provided a compositionfor sealing a wound, comprising a matrix as described herein. Accordingto at least some embodiments, there is provided a use of the compositionfor sealing suture or staple lines in a tissue.

According to at least some embodiments, there is provided a compositionfor a vehicle for localized drug delivery, comprising a matrix asdescribed herein. According to at least some embodiments, there isprovided a composition for tissue engineering, comprising a matrix asdescribed herein, adapted as an injectable scaffold.

According to at least some embodiments, there is provided a method ofmodifying a composition, comprising: providing a modified enzyme havinga cross-linkable functional group and a protein having at least onemoiety cross-linkable by said modified enzyme; mixing said modifiedenzyme and said protein, wherein said modified enzyme cross-links saidprotein and is also cross-linked to said protein through saidcross-linkable functional group.

Non-limiting examples of direct crosslinking enzymes, which directlycrosslink substrate groups on polymer strands, include transglutaminasesand oxidative enzymes. Examples of transglutaminases include microbialtransglutaminase (mTG), tissue transglutaminase (tTG), and Factor XIII.These enzymes can be from either natural or recombinant sources.Glutamine and lysine amino acids in the polymer strands are substratesfor transglutaminase crosslinking.

Non-limiting examples of oxidative enzymes are tyrosinase, laccase, andperoxidase. These enzymes crosslink polymers by quinone formation(tyrosinase) or free radical formation (laccase, peroxidase). Thequinones and the free radicals then interact with each other or withother amino acids or phenolic acids to crosslink the polymers. Thecrosslinkable substrates for these enzymes may be any proteins whichcontain tyrosine or other aromatic amino acids. The substrates may alsobe carbohydrates which contain phenolic acids such as freulic acid. Suchcarbohydrates may be arabinoxylan or pectin, for example.

Synthetic or partially synthetic polymers with one or more suitablefunctional groups could also serve as cross-linkable substrates for anyof the above enzymes.

In another embodiment of the present invention, a combination of enzymesis used.

“Polymer strands” or “Polymer chains” as defined herein refers to thesubstrate polymer for enzyme crosslinking, which according to at leastsome embodiments of the present invention, preferably belongs to one ofthe below categories (as non-limiting examples only and without wishingto provide a closed list):

-   -   1) Any polymer with substrate groups that are naturally        crosslinkable by the enzyme and that is itself naturally        crosslinkable by the enzyme. For example, in the case of        transglutaminases, this would include protein or polypeptides        such as gelatin, collagen, and casein which are naturally        crosslinkable by the enzyme.    -   2) Polymers which contain substrate groups crosslinkable by the        enzyme but which are not naturally crosslinkable by the enzyme        as a result of their structure. In such cases, the polymer        structure must be modified prior to enzyme crosslinking. For        example, in the case of transglutaminases, this would include        proteins, such as albumin or lactoglobulin, which are not        natural substrates for the enzyme because they have a globular        structure which hinders the access of the enzyme. These can be        made into substrates by partially denaturing the protein using        reducing agents, denaturing agents or heat.    -   3) Polymers, natural or synthetic, that are not substrates for        enzyme crosslinking but that have been modified with peptides or        functional groups which are substrates of the enzyme, thus        rendering the modified polymer crosslinkable by the enzyme.

Non-limiting examples of such polymers include any suitable type ofprotein, which may for example optionally comprise gelatin as notedabove. Gelatin may optionally comprise any type of gelatin whichcomprises protein that is known in the art, preferably including but notlimited to gelatin obtained by partial hydrolysis of animal tissueand/or collagen obtained from animal tissue, including but not limitedto animal skin, connective tissue (including but not limited toligaments, cartilage and the like), antlers or horns and the like,and/or bones, and/or fish scales and/or bones or other components;and/or a recombinant gelatin produced using bacterial, yeast, animal,insect, or plant systems or any type of cell culture.

According to preferred embodiments of the present invention, gelatinfrom animal origins preferably comprises gelatin from mammalian originsand more preferably comprises one or more of pork skins, pork and cattlebones, or split cattle hides, or any other pig or bovine source. Morepreferably, such gelatin comprises porcine gelatin since it has a lowerrate of anaphylaxis. Gelatin from animal origins may optionally be oftype A (Acid Treated) or of type B (Alkaline Treated), though it ispreferably type A.

Preferably, gelatin from animal origins comprises gelatin obtainedduring the first extraction, which is generally performed at lowertemperatures (50-60° C., although this exact temperature range is notnecessarily a limitation). Gelatin produced in this manner will be inthe range of 250-300 bloom and has a high molecular weight of at leastabout 95-100 kDa. Preferably, 275-300 bloom gelatin is used.

A non-limiting example of a producer of such gelatins is PB Gelatins(Tessenderlo Group, Belgium).

According to some embodiments of the present invention, gelatin fromanimal origins optionally comprises gelatin from fish. Optionally anytype of fish may be used, preferably a cold water variety of fish suchas carp, cod, or pike, or tuna. The pH of this gelatin (measured in a10% solution) preferably ranges from 4-6.

Cold water fish gelatin forms a solution in water at 10° C. and thus allcold water fish gelatin are considered to be 0 bloom. For the presentinvention, a high molecular weight cold water fish gelatin is optionallyand preferably used, more preferably including an average molecularweight of at least about 95-115 kDa. This is equivalent to the molecularweight of a 250-300 bloom animal gelatin. Cold water fish gelatinundergoes thermoreversible gelation at much lower temperatures thananimal gelatin as a result of its lower levels of proline andhydroxyproline. Per 1000 amino acid residues, cold water fish gelatinhas 100-130 proline and 50-75 hydroxyproline groups as compared to135-145 proline and 90-100 hydroxyproline in animal gelatins (Haug L T,Draget K I, Smidsrod O. (2004). Food Hydrocolloids. 18:203-213).

A non-limiting example of a producer of such a gelatin is NorlandProducts (Cranbury, N.J.).

In some embodiments of the present invention, low endotoxicity gelatinis used to form the gelatin solution component of the gelatin-mTGcomposition. Such a gelatin is available commercially from supplierssuch as Gelita™ (Eberbach, Germany). Low endotoxicity gelatin is definedas gelatin with less than 1000 endotoxicity units (EU) per gram. Morepreferably, gelatin of endotoxicity less than 500 EU/gram is used.

For very high sensitivity applications, such as with materials that willcome into contact with either the spine or the brain, gelatin withendotoxicity of less than 100 EU/gram is preferred, gelatin with lessthan 50 EU/g is more preferred. Gelatin with endotoxicity less than 10EU/g is very expensive but could also be used as part of at least someembodiments of the present invention in sensitive applications.

According to some embodiments of the present invention, type I, type II,or any other type of hydrolyzed or non-hydrolyzed collagen replacesgelatin as the protein matter being cross-linked. Various types ofcollagen have demonstrated the ability to form thermally stablemTG-crosslinked gels.

According to some embodiments of the present invention, a recombinanthuman gelatin is used. Such a gelatin is available commercially fromsuppliers such as Fibrogen™ (San Francisco, Calif.). Recombinant gelatinis preferably at least about 90% pure and is more preferably at leastabout 95% pure. Some recombinant gelatins are non-gelling at 10° C. andthus are considered to be 0 bloom. For some embodiments of the presentinvention, a high molecular weight recombinant gelatin is preferablyused, more preferably including a molecular weight of at least about95-100 kDa.

As noted above, the cross-linkable protein preferably comprises gelatinbut may also, additionally or alternatively, comprise another type ofprotein. According to some embodiments of the present invention, theprotein is also a substrate for transglutaminase, and preferablyfeatures appropriate transglutaminase-specific polypeptide and polymersequences. These proteins may optionally include but are not limited tosynthesized polymer sequences that independently have the properties toform a bioadhesive or polymers that have been more preferably modifiedwith transglutaminase-specific substrates that enhance the ability ofthe material to be cross-linked by transglutaminase. Non-limitingexamples of each of these types of materials are described below.

Synthesized polypeptide and polymer sequences with an appropriatetransglutaminase target for cross-linking have been developed that havetransition points preferably from about 20 to about 40° C. Preferredphysical characteristics include but are not limited to the ability tobind tissue and the ability to form fibers Like gelling type gelatins(described above), these polypeptides may optionally be used incompositions that also feature one or more substances that lower theirtransition point.

Non-limiting examples of such peptides are described in U.S. Pat. Nos.5,428,014 and 5,939,385, both filed by ZymoGenetics Inc, both of whichare hereby incorporated by reference as if fully set forth herein. Bothpatents describe biocompatible, bioadhesive, transglutaminasecross-linkable polypeptides wherein transglutaminase is known tocatalyze an acyl-transfer reaction between the γ-carboxamide group ofprotein-bound glutaminyl residues and the ε-amino group of Lys residues,resulting in the formation of ε-(γ-glutamyl) lysine isopeptide bonds.

According to some embodiments, the resultant composition is used as avehicle for localized drug delivery.

According to some embodiments, the resultant composition is aninjectable scaffold for tissue engineering.

According to some embodiments, the composition is a hemostaticcomposition. According to some embodiments, the composition is a bodyfluid sealing composition.

The compositions of the present invention preferably provide rapidhemostasis, thereby minimizing blood loss following injury or surgery.

“Wound” as used herein refers to any damage to any tissue of a patientthat results in the loss of blood from the circulatory system or theloss of any other bodily fluid from its physiological pathway, such asany type of vessel. The tissue can be an internal tissue, such as anorgan or blood vessel, or an external tissue, such as the skin. The lossof blood or bodily fluid can be internal, such as from a ruptured organ,or external, such as from a laceration. A wound can be in a soft tissue,such as an organ, or in hard tissue, such as bone. The damage may havebeen caused by any agent or source, including traumatic injury,infection or surgical intervention. The damage can be life-threateningor non-life-threatening.

Surgical wound closure is currently achieved by sutures and staples thatfacilitate healing by pulling tissues together. However, very often theyfail to produce the adequate seal necessary to prevent fluid leakage.Thus, there is a large, unmet medical need for devices and methods toprevent leakage following surgery, including leaks that frequently occuralong staple and suture lines. Such devices and methods are needed as anadjunct to sutures or staples to achieve hemostasis or otherfluid-stasis in peripheral vascular reconstructions, durareconstructions, thoracic, cardiovascular, lung, neurological, andgastrointestinal surgeries. Most high-pressure hemostatic devicescurrently on the market are nominally, if at all adhesive. Thus, thecompositions of the present invention, according to at least someembodiments, overcome these drawbacks and may optionally be used forhemostasis.

As used herein, “about” means plus or minus approximately ten percent ofthe indicated value.

Other features and advantages of the various embodiments of theinvention will be apparent from the following detailed description, andfrom the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIG. 1: Effect of reaction pH and activated PEG concentration onPEGylation products size and distribution;

FIG. 2: Effect of reaction time and pH on size and distribution ofPEGylation products;

FIG. 3: SDS-analysis of PEGylated mTG using various concentrations ofPEG-NHS (2 kD);

FIG. 4: Elution of mTG and PEGylated mTG from the same crosslinkedgelatin gel;

FIG. 5: Elution of mTG (left) and PEGylated mTG (right) from differentcrosslinked gelatin gels;

FIG. 6: Burst pressure values for gelatin sealant made withnon-PEGylated mTG and 2 types of PEGylated mTG;

FIG. 7: SDS-PAGE analysis of conjugation products between mTG anddextran;

FIG. 8: SDS-PAGE analysis of PEGylation products of horseradishperoxidase (HRP);

FIG. 9: SDS-PAGE analysis of PEGylation products of mTG, using variousreactions conditions, the gel demonstrates various degrees ofPEGylation;

FIG. 10: SDS-PAGE analysis of PEGylation products of mTG, where thereactive PEG is a bifunctional 10 kD PEG-NHS;

FIG. 11 shows mass to charge spectrum of a typical batch of PEGylatedmTG acquired by MALDI-TOF mass spectrometer; and

FIG. 12 shows SDS-PAGE analysis of PEGylation products of mTG where PEGreagent to amine ratio is kept constant but reactant concentration isvaried.

DETAILED DESCRIPTION OF INVENTION

The section headings that follow are provided for ease of descriptiononly. It is to be understood that they are not intended to be limitingin any manner. Also, unless otherwise defined, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, suitable methods and materials are described below. In caseof conflict, the patent specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

Increased Perceived Volume of Enzyme Crosslinker in Hydrogel

It was found that, in addition to the viscosity of the enzyme-containingpolymer solution and the crosslinking density of the partially crosslinked solution (availability of reactive groups), the catalytic rate ofa crosslinking enzyme within a crosslinked matrix can also be controlledthrough control of the perceived volume of the enzyme molecule.

According to at least some embodiments of the present invention, suchcontrol can optionally and preferably lead to reduced catalytic rate ofcrosslinking as the matrix approaches a desired mechanical state, byincreasing the perceived volume of the enzyme molecule prior toinitiation of the crosslinking reaction or during the reaction itself.In this manner, the solidifying matrix traps the size-enhanced enzyme atthe desired crosslinking density state and further crosslinking isprevented. Perceived enzyme volume is a function of enzyme molecularweight and hydrodynamic volume, among other factors.

The ultimate extent of crosslinking within a crosslinked matrix can belimited by engineering the enzyme molecules, the matrix material, thecrosslinking environment, or some combination of these factors toincrease the perceived volume of the enzyme molecules within thecrosslinked matrix as the matrix is formed. Without wishing to belimited by a single hypothesis, it is possible that increased perceivedenzyme volume results in reduced mobility of the enzyme in thecrosslinked matrix. Reducing enzyme mobility to control ultimatecrosslinking density is most effective when the enzyme moleculesmaintain mobility at the early crosslinking reaction stages when thesolution viscosity is still low, but lose mobility as crosslinkingprogresses to increase the solution viscosity, and lose mobility moreseverely after the initial solid matrix or hydrogel has been formed.Naturally, the precise levels of enzyme mobility within the matrixshould be regulated to achieve the crosslinking profile and extentdesirable for a particular application.

Without wishing to be limited by a single hypothesis, an enzyme with anincreased size or increased hydrodynamic volume has a lower diffusioncoefficient or mobility in the crosslinked matrix than the non-modifiedenzyme, resulting in a more limited access to crosslinkable substrates .

Enzyme Molecules with Increased Size and/or Hydrodynamic Volume

A preferred method of reducing the mobility of enzyme molecules in acrosslinked matrix is increasing the effective size of the enzymemolecules. This can be accomplished by increasing the enzyme moleculemolecular weight (MW), hydrodynamic volume, or both MW and hydrodynamicvolume. This is a preferred method because it should not affect thestructural composition of the crosslinked matrix.

To be effective for the herein described embodiments of the presentinvention, enzyme molecule size is preferably increased in a manner thatdoes not eliminate enzyme activity or its ability to crosslink thedesired polymer substrate into a solid matrix or hydrogel. The enzymealso preferably retains sufficient activity to form the matrix within anappropriate amount of time. Furthermore, the size-enhanced enzymemolecule also preferably retains sufficient mobility within thecrosslinked matrix to catalyze the desired degree of crosslinking priorto ceasing mobility within the matrix.

A number of methods have been identified for increasing enzyme moleculesize in crosslinked matrices or hydrogels:

-   -   1. Cross link the enzyme to itself (intermolecular crosslinking)        in order to from soluble multi-unit conjugates. An example of        this is described in example 18, below.    -   2. Covalent binding (immobilization) of the enzyme on a carrier:        -   I. Immobilization to a soluble protein, for example albumin;            (Allen T M et al, 1985, JPET 234: 250-254,            alpha-Glucosidase-albumin conjugates: effect of chronic            administration in mice)        -   II. Immobilization on a soluble polymer. Preferably, the            polymer carrier is larger than the enzyme, where one or more            enzyme molecules are immobilized on each molecule of the            polymer. It is also possible that a single enzyme molecule            will bind to more than one polymer molecule via two or more            attachment sites. The carrier may be natural, synthetic or            semi-synthetic. Many such applications were developed in            order to increase the in vivo stability of enzymes or to            reduce immunogenicity. One such family of polymers is            cellulose ethers, including but not limited to carboxymethyl            cellulose, hydroxypropyl methylcellulose, hydroxyethyl            cellulose, methyl cellulose and others. Such immobilization            has previously been accomplished with enzymes such as            trypsin (Villaonga et al, 2000, Journal of Molecular            Catalysis B: 10, 483-490 Enzymatic Preparation and            functional properties of trypsin modified by            carboxymethylcellulose) and lysozyme (Chen S H et al, 2003,            Enzyme and Microbial Technology 33, 643-649, Reversible            immobilization of lysozyme via coupling to reversibly            soluble polymer), though such enzyme immobilization has            never previously been used to affect mechanical properties            of enzyme-crosslinked hydrogels or matrices.        -   III. Binding to a glycosaminoglycan (GAG), including but not            limited to chondroitin sulfate, dermatan sulfate, keratan            sulfate, heparin, heparan sulfate, and hyaluronic acid. As            above, such binding has been accomplished            (Luchter-Wasylewska E et al., 1991, Biotechnology and            applied biochemistry 13: 36-47, Stabilization of human            prostatic acid phosphatase by coupling with chondroitin            sulfate), though never used to affect mechanical properties            of enzyme-crosslinked hydrogels or matrices.        -   IV. Enzymes can also be coupled to polysaccharides, such as            dextran and starch derivatives such as hydroxyethyl starch.            An example of this can be seen in example 13 where an enzyme            was coupled to oxidized dextran.    -   3. Addition of one or more moieties to a single enzyme molecule        through covalent modification(s). Often, but not always, the        said moiety is smaller than the enzyme. An example for such a        modification is PEGylation of the crosslinking enzyme, as        extensively described below in multiple examples.    -   4. Other types of covalent binding. For example, by grafting        biotin molecules on the surface of the enzyme (biotinylation)        and immobilizing the biotinylated enzyme on avidin or        streptavidin containing molecules or polymers. The carrier may        be a non crosslinkable soluble polymer whose function is to        capture the crosslinking enzyme before or during the        crosslinking reaction. Alternatively, the capturing groups, e.g.        avidin or streptavidin may be grafted on the crosslinkable        polymer itself, resulting in gradual immobilization of the        crosslinking enzyme during the crosslinking reaction on the        crosslinkable polymer.    -   5. Non-covalent binding of the enzyme to a carrier or polymer.        For example, electrostatic interactions between the enzyme and        the carrier or polymer may provide a stable but non-covalent        bond when the net charge of the enzyme has an opposite sign to        the net charge of the carrier.        Technologies Related to Increasing the Size of Enzyme Molecules

Though increasing the size of enzymes has been previously disclosed onseveral occasions, it has never been considered in the context offorming and/or controlling the formation of enzyme crosslinked matricesor hydrogels. Application of size-increased enzymes in crosslinkedmatrices is entirely novel as the crosslinking reactivity of sizeincreased enzymes in such matrices has not previously been characterizedto any degree. Furthermore, the inventors of the present invention havesurprisingly demonstrated that isolated enzyme activity of size-enhancedenzyme, as tested in a colorimetric enzyme activity assay, is distinctlydifferent from the crosslinking activity of size-enhanced enzyme inhydrogel formation as indicated by gelation rate. For example, Example 5describes a comparison of enzyme-catalyzed gelation rate to enzymeactivity values measured using a colorimetric assay. PEGylation isdescribed in this Example as a non-limiting, illustrative method forincreasing enzyme size.

PEGylation is the covalent attachment of polyethylene glycol (PEG)molecules to enzyme molecules and is a preferred method of increasingenzyme molecule size. The operation of adding such one or more PEGmolecules is known as PEGylation.

PEG is a desirable material for use in increasing enzyme size as it isbio-inert and has also demonstrated the ability to limit the immunogenicresponse to PEGylated implanted or injected molecules. Although it isnot known whether PEGylation of enzymes as described herein also causessuch immunogenic masking (limited immunogenic response), without wishingto be limited by a single hypothesis, it is possible that in factPEGylation of the enzyme does limit the immunogenic response to theenzyme and also possibly, by extension, to the crosslinked matrix.

One method of accomplishing enzyme PEGylation is by reacting the enzymewith activated metoxyl PEG (mPEG) that react with amine groups on theenzyme (amine PEGylation). Non-limiting examples of activated mPEGinclude succinimidyl (NHS) esters of mPEG (mPEG-succinate-NHS,mPEG-glutarate-NHS, mPEG-valerate-NHS, mPEG-carbonate-NHS,mPEG-carboxymethyl-NHS, mPEG-propionate-NHS, mPEG-carboxypentyl-NHS),mPEG-nitrophenylcarbonate, mPEG-propylaldehyde, mPEG-Tosylate,mPEG-carbonylimidazole, mPEG-isocyanate, mPEG-epoxide.

The activated mPEGs can be those that react with thiol groups on theenzymes (thiol PEGylation).

The activated PEGs may be monofunctional, heterobifunctional orhomobifunctional.

The activated PEGs may be branched PEGs or multi-arm PEGs.

The size of the activated PEG may range from 1000 dalton to 40,000dalton

The molar ratio of the activated PEG to lysine groups on the enzyme isfrom 0.1:1 to 100:1 and preferably 0.5:1 to 10:1

Preferably, the pH of the PEGylation reaction is 7-9. More preferablythe pH of the reaction is 7.5 -8.5.

According to a preferred embodiment, the PEGylated enzyme may be furtherpurified from non-reacted enzyme or in order to reduce the size range ofthe PEGylation products. The purification may be done usingsize-exclusion chromatography.

Alternatively, or in addition, the purification may be done using ionicchromatography, such as SP-sephrose, Q-sepharose, SM-sepharose orDEAE-sepharose. Alternatively, or in addition, purification fromnon-reacted enzyme may also be done using dialysis, ultrafiltration orammonium sulfate fractionation.

Various examples provided below describe the use of PEGylation oftransglutaminases for control of cross-linked hydrogel formation.Example 1 describes PEGylation reaction of mTG with PEG-NHS (5 kD). Thesize and distribution of PEGylation products is dependent on the PEG tomTG ratio as well as the pH of the reaction.

Example 2 describes PEGylation reaction of mTG with PEG-NHS (5 kD). Thesize and distribution of PEGylation products is dependent on theduration and pH of the reaction.

Example 3 describes PEGylation reaction of mTG with PEG-NHS (2 kD). Thesize and distribution of PEGylation products is dependent on the PEG tomTG ratio.

Example 4 describes a TNBS assay for the determination of the PEGylationextent of various preparations of PEGylated mTG (5 kD PEG). The resultssuggest that the extent of PEGylation depends on the activated PEG:mTGratio in the reaction.

Example 5 describes assays for the determination of activity ofPEGylated mTG. The results suggest that PEGylated mTG retains most itsactivity towards small substrates, such as hydroxylamine and CBZ-Gln-Glybut loses a significant portion of its activity towards largersubstrates such as gelatin.

Examples 6 and 7 describe SDS-PAGE analysis of elution profile of mTGand PEGylated mTG from crosslinked gelatin gels. The results suggestthat the PEGylated mTG elutes from the gel more slowly and to a lesserextent than non-PEGylated mTG, possibly due to its larger size orhydrodynamic volume.

Example 8 describes the measurement of activity of mTG that has elutedfrom crosslinked gelatin gels. The results suggest that non-PEGylatedmTG which is eluted from crosslinked gelatin gels retains most of itsactivity (86% of maximal calculated activity).

Example 9 describes the mechanical testing of gelatin gels crosslinkedwith PEGylated or non-PEGylated mTG. The results demonstrate thatgelatin gels crosslinked with PEGylated mTG are stronger andconsiderably more flexible than gels cross-linked with non-PEGylatedmTG.

Example 10 describes burst pressure testing of various gelatin sealantformulations. The results suggest that gelatin sealants made withPEGylated mTG demonstrate burst pressures results which are comparableto those of sealants made with non-PEGylated mTG.

Example 11 describes use of sealant for staple line reinforcement for invivo porcine model.

Example 12 describes the effect of non-covalent binding of cross-linkingenzyme to insoluble carrier. Example 13 describes the effect of enzymemodification with oxidized dextran.

Example 14 demonstrates that modification of Horseradish Peroxidase(another crosslinking enzyme) by PEGylation can modify matrices formedby peroxidase crosslinking.

Example 15 demonstrates the effect of partial PEGylation of thecross-linking enzyme.

Example 16 demonstrates that free PEG (PEG molecule placed in solutionwith the crosslinking enzyme, but not covalently bound to the enzyme)has no effect on gelation.

Example 17 illustrates the effect of various mixtures of modified enzymemixed with non-modified enzyme on gelation.

Example 18 demonstrates the effect of bi-functional PEG-enzyme bridgeson gelation.

Example 19 relates to mass spectrometry analysis of PEGylated mTG(microbial transglutaminase).

Example 20 describes PEGylation of mTG at a fixed PEG to amine ratiowith various concentrations of reactants, demonstrating the large effectof total reactant concentration on the extent of PEGylation.

Surprisingly it was found in these Examples that while PEGylationreduced the rate at which microbial transglutaminase (mTG) crosslinkedgelatin, it did not decrease its activity in the hydroxamate assay,which is a gold standard activity assay for transglutaminases. Theseresults contradict the background art teachings which indicated thatsize-enhanced enzyme might be undesirable for use in hydrogel formationas it might have significantly lower efficacy in causing hydrogelformation.

It should be noted that TGases (transglutaminases) are sometimesmentioned in the context of PEGylation in the background art; however,these references teach the use of TGase as a tool for enabling orenhancing site specific PEGylation of other proteins (rather than as asubstrate for PEGylation) by catalyzing the transglutamination reactionof glutamyl residues on the said proteins with a primary amine groupattached to the said PEG molecules. However, such background art doesnot teach or suggest PEGylation of TGases themselves in order to alteror control their crosslinking activity or to alter or control themechanical properties of hydrogel matrices crosslinked by these enzymes.

Reduced Mobility of Crosslinking Enzyme by Coupling onto CrosslinkedMatrix

In another embodiment of reducing enzyme mobility in a crosslinkedmatrix, the enzyme undergoes a binding reaction to the crosslinkedmatrix itself simultaneous to catalyzing the crosslinking reaction. Asthe enzyme moves through the polymer solution to crosslink the polymersin a matrix, it is gradually bound to the polymers themselves and thusimmobilized in the matrix. For example, biotinylated enzyme can be mixedwith a crosslinkable polymer component containing avidin or streptavidincoated polymer. U.S. Pat. No. 6,046,024 (Method of producing a fibrinmonomer using a biotinylated enzyme and immobilized avidin) describes amethod of capturing biotinylated thrombin from fibrinogen solution byadding avidin-modified agarose. Though in this case, the agarose was notsoluble, it is possible to bind avidin or streptavidin to water solublepolymer as well as described by U.S. Pat. No. 5,026,785 (Avidin andstreptavidin modified water-soluble polymers such as polyacrylamide, andthe use thereof in the construction of soluble multivalentmacromolecular conjugates). Biotinylation of transglutaminase andsubsequent adsorption to avidin-treated surfaces has been shown to befeasible (Huang X L et al, J. Agric. Food Chem., 1995, 43 (4), pp895-901). Alternatively, the crosslinking enzyme may be covalently boundto avidin or streptavidin and the conjugate added to the crosslinkingreaction which contains a biotinylated polymer. The biotinylated may bethe crosslinkable polymer itself, e.g. gelatin, or a non-crosslinkableco-polymer such as dextran. Dextran-biotin conjugates of molecularweights of up to 500,000 dalton are available from commercial sources.

Reduced Mobility of Crosslinking Enzyme by Electrostatic Interactions inCrosslinked Matrix

In another embodiment of the present invention, enzyme mobility isreduced through reversible binding based on electrostatic interactionsbetween the enzyme and a polymer carrier in which the net charge of theenzyme has an opposite sign to the net charge of the carrier. The enzymemay be pre-incubated with the carrier and added to the crosslinkingreaction or it may be bound to the carrier during the crosslinkingreaction. For example, if the crosslinking enzyme is positively chargedat neutral pH it may be electrostatically bound to a negatively chargedcarrier, for example carboxymethyl cellulose (CMC). The enzyme may beincubated with CMC to allow binding and then the complex added to thecrosslinking reaction, or the enzyme and CMC are added separately. Inthe latter case the enzyme will bind the CMC gradually during thecrosslinking reaction. It is also possible to bind the enzyme to thecrosslinkable polymer strands themselves during the crosslinkingreaction, provided that the crosslinkable polymer bears an opposite signcharge relative to the crosslinking enzyme. Alternatively, theisoelectric point (pI) of the crosslinking enzyme can be shifted suchthat the enzyme acquires an opposite sign charge than that of thecrosslinkable polymer or carrier.

In another embodiment, the crosslinking enzyme is modified in such a waythat its isoelectric point (pI) is changed to result in a different netcharge on the enzyme at a given pH. Examples of ways to reduce the pI ofthe enzyme are to modify the ε-amino group of lysines by processes suchas but not limited to succinylation (with succinic anhydride),acetylation (with acetic anhydride), carbamylation (with cyanate),reductive alkylation (aldehydes) and treatment with maleic anhydride.This results in decrease in the positive net charge on the protein by upto one charge unit per modified amino acid (except for succinylationwhich decreases the positive net charge by up to two charge units) anddecrease in the pI. Conversely, side chains containing carboxylic acidssuch as glutamic and aspartic acid may be modified in order to decreasethe number of negative charges on the protein and as a result increasethe pI. For example it is possible to treat the enzyme with EDC1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide) and ethylene diamine(EDA). EDC activates the carboxylic acid groups and an amide bond isformed between them and EDA. The result is an increase in the positivenet charge of the protein and in the pI.

The release of proteins from hydrogels has been linked to electrostaticattraction and repulsion forces between the hydrogel polymer chains andthe entrapped protein. It has been suggested that electrostaticrepulsion forces increase the diffusion coefficient of the entrappedprotein and conversely, electrostatic attraction forces decrease thediffusion coefficient in protein release experiments from recombinantgelatin matrix (Marc Sutter-Juergen Siepmann, Wim E. Hennink and WimJiskoot, Recombinant gelatin hydrogels for the sustained release ofproteins, Journal of Controlled Release Volume119, Issue 3, 22 June2007, Pages 301-312)

Changing the pI of the hydrogel polymer chain itself has been suggestedas a way to control the release of proteins from that hydrogel. However,the background art involved manipulating electrostatic interactionsbetween proteins entrapped within a hydrogel and the hydrogel chains areconcerned with methods of controlling the release rate of thetherapeutic proteins from the hydrogel, where the proteins are notthemselves involved in the formation of the hydrogel. For at least someembodiments of the present invention, the electrostatic interactions aremodified to improve the hydrogel mechanical properties, which may berelated to mobility and diffusion coefficient of the enzyme in thehydrogel matrix that the enzyme is crosslinking.

Changing the pI of the entrapped crosslinking enzyme is therefore anovel approach to prevent over crosslinking because the diffusion ormobility of the cross linking enzyme in the cross linkable matrix isseverely restricted by modification of the pI of the entrapped enzymerather than of the polymeric hydrogel.

EXAMPLE 1 Effect of Reaction pH, and PEG: mTG Ratio on Size andDistribution of PEGylation Products

Materials:

-   Activated PEG: mPEG-glutarate-NHS 5 kDa (SunBright ME-050GS, NOF    corporation, Japan)-   mTG: Ajinimoto activa 10% further purified using SP-sepharose ion    exchange chromatography. Activity: 604 units/ml in 0.2 M sodium    citrate pH 6-   sodium citrate, Hepes , SDS and beta mercaptoethanol were from Sigma    Aldrich.-   30% Acrylamide/Bis 29:1 and Bio-Safe Coomassie G-250 stain were from    Bio-Rad.-   Molecular weight marker was Precision Plus Dual Color (Bio-Rad)

1 unit of mTG activity catalyzes the formation of 1.0 μmol ofhydroxamate per min from N-CBZ-Gln-Gly and hydroxylamine at pH 6.0 at37° C. A set of reactions was set up, each with a volume of 0.2 ml. Allreactions contained 15 u/ml mTG, the approprtiate reaction buffer—either90 mM sodium citarte, pH 6 or 100 mM Hepes pH 7, and various amounts ofactivated PEG. The PEG-NHS reacts with primary amines in proteins, theepsilon-amine on side chains of lysine residues as well as the aminoterminus of proteins. The ratios of PEG to lysine residues in thereaction mix is described in detail below,

The reactions were incubated at 37° C. for 1:36 hr and then glycine wasadded to a final concentration of 110 mM in order to neutralize theexcess of activated PEG molecules that have not reacted with the enzyme.

Samples from each reaction were denatured by heating at 90° C. in thepresence of SDS and beta mercaptoethanol and were analyzed usingSDS-PAGE (8% resolving gel, 4% stacking gel, Mini-Proteanelectrophoresis system, BioRad). To visualize the proteins the gel wasstained with Bio-Safe Coomassie G-250 stain followed by destaining withwater. The gel was scanned with CanoScan 8800 F scanner and the image isshown in FIG. 1, showing the effect of reaction pH and activated PEGconcentration on PEGylation products size and distribution. Laneassignments were as follows:

-   Lane 1: mTG (control)-   Lane 2: Molecular size marker (from top to bottom: 250 kD, 150 kD,    100 kD, 75 kD, 50 kD, 37 kD, 25 kD)-   Lane 3: 53.3 mg/ml activated PEG; 90 mM Na citrate pH 6; PEG to    lysine ratio 9.15-   Lane 4: 26.6 mg/ml activated PEG; 90 mM Na citrate pH 6; PEG to    lysine ratio 4.59-   Lane 5: 13.3 mg/ml activated PEG; 90 mM Na citrate pH 6; PEG to    lysine ratio 2.30-   Lane 6: 53.3 mg/ml activated PEG; 100 mM Hepes pH 7; PEG to lysine    ratio 9.15-   Lane 7: 26.6 mg/ml activated PEG; 100 mM Hepes pH 7; PEG to lysine    ratio 4.59-   Lane 8: 13.3 mg/ml activated PEG; 100 mM Hepes pH 7 PEG to lysine    ratio 2.30

As can be seen from FIG. 1, larger amounts of PEG and increased pHresulted in enzyme having an increased apparent molecular weight on thegel.

EXAMPLE 2 Effect of Reaction pH and Duration on Size and Distribution ofPEGylation Products

All reactions contained 15 u/ml mTG.

Materials:

-   Activated PEG: mPEG-glutarate-NHS 5 kDa (SunBright ME-050GS, NOF    corporation, Japan)-   mTG: Ajinimoto activa 10% further purified using SP-sepharose ion    exchange chromatography. Activity: 604 units/ml in 0.2 M sodium    citrate pH 6-   sodium citrate, Hepes , SDS and beta mercaptoethanol were from Sigma    Aldrich.-   30% Acrylamide/Bis 29:1 and Bio-Safe Coomassie G-250 stain were from    Bio-Rad.-   Molecular weight marker was Precision Plus Dual Color (Bio-Rad)

1 unit of mTG activity will catalyze the formation of 1.0 μmol ofhydroxamate per min from N-CBZ-Gln-Gly and hydroxylamine at pH 6.0 at37° C.

A set of reactions was set up, each with a volume of 0.2 ml, Allreactions contained 15 u/ml mTG, the approprtiate reaction buffer-either 100 mM Hepes, pH 7 or 100 mM Hepes pH 8, and 25 mg/ml PEG-NHS.The ratio of PEG to lysine residues in the reaction mix was 4.59.

The reactions were incubated at room temperature for 2 hr. Samples weretaken at various time points as described below and glycine was added toa final concentration of 110 mM in order to neutralize the excess ofactivated PEG molecules that have not reacted with the enzyme.

Samples from each reaction were denatured by heating at 90° C. in thepresence of SDS and beta mercaptoethanol and were analyzed usingSDS-PAGE (8% resolving gel, 4% stacking gel, Mini-Proteanelectrophoresis system, BioRad). To visualize the proteins the gel wasstained with Bio-Safe Coomassie G-250 stain followed by destaining withwater. The gel was scanned with CanoScan 8800F scanner and the image isshown in FIG. 2, demonstrating the effect of reaction time and pH onsize and distribution of PEGylation products. Lane assignments are asfollows:

-   Lane 1: 25 mg/ml activated PEG; 100 mM Hepes pH 8; 15 min reaction    time-   Lane 2: 25 mg/ml activated PEG; 100 mM Hepes pH 8; 30 min reaction    time-   Lane 3: 25 mg/ml activated PEG; 100 mM Hepes pH 8; 60 min reaction    time-   Lane 4: 25 mg/ml activated PEG; 100 mM Hepes pH 8; 120 min reaction    time-   Lane 5: Molecular size marker (from top to bottom: 250 kD, 150 kD,    100 kD, 75 kD, 50 kD, 37 kD, 25 kD)-   Lane 6: 25 mg/ml activated PEG; 100 mM Hepes pH 7; 15 min reaction    time-   Lane 7: 25 mg/ml activated PEG; 100 mM Hepes pH 7; 30 min reaction    time-   Lane 8: 25 mg/ml activated PEG; 100 mM Hepes pH 7; 60 min reaction    time-   Lane 9: 25 mg/ml activated PEG; 100 mM Hepes pH 7; 120 min reaction    time

As shown in FIG. 2, increased reaction time and increased pH resulted inenzyme having an increased apparent molecular weight on the gel.

EXAMPLE 3 PEGylation of mTG with PEG-NHS (2 kD): effect of PEG: mTGRatio on size and distribution of PEGylation products

Materials:

-   Activated PEG: mPEG-glutarate-NHS 2 kDa (SunBright ME-020CS, NOF    corporation, Japan)-   mTG: Ajinimoto activa 10% further purified using SP-sepharose ion    exchange chromatography. Activity: 604 units/ml in 0.2 M sodium    citrate pH 6 sodium citrate, Hepes , SDS and beta mercaptoethanol    were from Sigma Aldrich. 30% Acrylamide/Bis 29:1 and Bio-Safe    Coomassie G-250 stain were from Bio-Rad.-   Molecular weight marker was Precision Plus Dual Color (Bio-Rad).

1 unit of mTG activity will catalyze the formation of 1.0 μmol ofhydroxamate per min from N-CBZ-Gln-Gly and hydroxylamine at pH 6.0 at37° C. Reactions (200 μl) contained 15 u/ml mTG, 100 mM Hepes, pH 8 andvarious concentrations of PEG NHS (2 kD). The reactions were incubatedat 37° C. for 2 hours, followed by addition of 10 μl 1.5 M glycine (71mM final concentration) in order to neutralize the PEG-NHS moleculesthat have not reacted with the enzyme. Samples from each reaction weredenatured by heating at 90° C. in the presence of SDS and betamercaptoethanol and were analyzed using SDS-PAGE (8% resolving gel, 4%stacking gel, Mini-Protean electrophoresis system, BioRad). To visualizethe proteins the gel was stained with Bio-Safe Coomassie G-250 stainfollowed by destaining with water. The gel was scanned with CanoScan8800F scanner and the image is shown in FIG. 3, demonstratingSDS-analysis of PEGylated mTG using various concentrations of PEG-NHS(2kD). Lane assignments are as follows:

-   Lane 1: Molecular size marker-   Lane 2:1.75 mg/ml PEG-NHS 2 kD; PEG to lysine ratio 0.74-   Lane 3:3.5 mg/ml; PEG-NHS 2 kD; PEG to lysine ratio 1.48-   Lane 4: 7 mg/ml; PEG-NHS 2 kD ; PEG to lysine ratio 2.97-   Lane 5: 14 mg/ml; PEG-NHS 2 kD ; PEG to lysine ratio 5.93-   Lane 6:28 mg/ml; PEG-NHS 2 kD ; PEG to lysine ratio 11.86-   Lane 7:56 mg/ml; PEG-NHS 2 kD ; PEG to lysine ratio 23.72

As shown in FIG. 3, increased amounts of PEG-NHS resulted in enzymehaving an increased apparent molecular weight on the gel.

EXAMPLE 4 TNBS Assay for Determining Extent of PEGylation

Materials:

-   Glycine and 5% TNBS solution (picrylsulfonic acid) were from Sigma    Aldrich Sodium bicarbonate was from Frutarom (Israel)-   Dilute TNBS solution was prepared by mixing 5% TNBS 1 in 500 in    bicarbonate buffer (pH 8.5)-   The spectrophotometer was Anthelie Advanced (Secomam)

For calibration curve, the following solutions of glycine were preparedin bicarbonate buffer (pH 8.5): 1 μg/ml, 2 μg/ml, 4 μg/ml, 8 μg/ml

0.5 ml of diluted TNBS solution was mixed with 1 ml of standard glycinesolution or sample. The mixture was incubated at 37° C. for 2 hours.Next, 0.5 ml of 10% SDS solution and 0.25 ml of 1 M HCL were added tostop the reaction. The solutions were transferred to a cuvette and theO.D. was read at 335 nm using a spectrophotometer.

The percentage of free NH₂ groups was determined for each PEGylated mTGbased on the calibration curve set up for glycine.

% PEGylation=100-% of free NH₂

Calculated average MW of PEGylated mTG: 38,000+(% PEGylation:100× 18×5000).

The results are shown in Table 1 below.

TABLE 1 Calculated PEG-NHS average MW (5 kD) conc. in PEG to of reactionlysine % of free NH₂ PEGylated mg/ml ratio groups % PEGylation mTG 3.50.59 38.8 61.2  93.08 kD 7 1.19 28.9 71.1 101.99 kD 14 2.37 20.8 79.2109.28 kD 28 4.75 13.9 86.1 115.49 kD 56 9.49 10.9 89.1 118.19 kD

The above table shows that increased PEGylation results in increasedapparent (calculated) molecular weight of mTG; furthermore, the degreeof PEGylation correlated with the reduction in the percentage of freelysine groups, indicating that PEGylation was occurring as expected onthe lysine groups.

EXAMPLE 5 Assays for Measuring the Activity of the PEGylated mTG

Materials:

-   Urea, Na citrate, Na Acetate and calcium chloride were from Sigma    Aldrich Gelatin (Pig skin Type A 275 bloom) was from Gelita

The PEGylation reaction (8 ml) contained 15 u/ml mTG, 100 mM HEPES (pH7) and 14 mg/ml PEG-NHS (5kD). The reaction conditions were similar tothose in lane 8 in FIG. 1.

The reaction incubated at 37° C. for 1:50 hours, followed by addition of0.4 ml 2.34 M glycine (100mM final concentration) in order to neutralizethe non-reacted activated PEG. After 15 minutes at room temperature thereaction mix was concentrated to 2 ml using Amicon Ultra-4 CentrifugalFilter Unit MWCO 30,000 (Millipore) and the reaction buffer changed to0.2 M sodium citrate. The concentrated PEGylated mTG is referred to as4×, while 2-fold and 4-fold dilutions of it in citrate buffer arereferred to as 2× and 1×, respectively.

Activity Assay Using Gelatin as a Substrate

-   0.5 ml of mTG was mixed with 1 ml of gelatin formulation (25%    gelatin, 3.8 M urea,-   0.15 M CaCl₂, 0.1 M Na acetate pH 6), incubated at 37° C. and the    gelation time was recorded. By definition, gelation time is the time    at which the liquid stops flowing when the reaction tube in    inverted.    Activity Assay Using the Hydroxamate Assay-   Reaction A 15 μl 1Xnon-PEGylated mTG (15 u/ml)+135 μl citrate buffer-   Reaction B 15 μl 1XPEGylated mTG+135 μl citrate buffer-   Reaction C 15 μl 2XPEGylated mTG+135 μl citrate buffer-   Reaction D 15 μl 4XPEGylated mTG+135 μl citrate buffer    1 mL of reaction cocktail was added to each of reaction A-D and the    mix was incubated at 37° C. for 10 minutes or 20 minutes. At each    time point, 0.23 ml of the reaction was added to a tube with 0.5 mL    TCA and 0.5 mL.    Hydroxamate Reaction Substrate Cocktail (20 ml, pH 6):-   240 mg CBZ-Glu-Gly (Sigma Aldrich)-   139 mg hydroxylamine hydrochloride (Sigma Aldrich)-   61.5 mg gluthatione reduced (Sigma Aldrich) 4 ml 0.2 M Na citrate    buffer pH 6-   Water to 20 ml

The results are shown in Table 2 below.

TABLE 2 Pre- Pegylation OD 525 in enzyme Gelation time hydroxamate assaySample tested activity with SLR 10 minutes 20 minutes 1X Non- 15 u/ml 4min 0.376 0.776 PEGylated mTG 1X PEGylated 15 u/ml 9 min 0.358 0.722 mTG2X PEGylated 30 u/ml 4.5-5 min    0.667 1.34 mTG 4X PEGylated 60 u/ml2.75 min   1.263 2.152 mTG

Table 2 above shows that PEGylation of the transglutaminase caused anincrease in gelation time at 1× PEGylation, but had little effect on theenzyme's activity in the hydroxamate assay (which occurs in freesolution, without a hydrogel being formed). Increased amounts ofPEGylation actually increased gelation time, presumably by reducing themobility of the enzyme required for collision with substrate moleculeswithin the forming crosslinked polymer network . Alternate explanationsare that PEGylation is conferring a structural alteration to theenzyme's active site such that it cannot accommodate the substrate asefficiently as non-PEGylated enzyme or that the one or more PEGmolecules inserted in the vicinity of the active site of the enzymecause steric hindrance to an approaching substrate molecule. It ispossible that the smaller size of substrate or the lack of crosslinkedpolymer network formation during the reaction in the hydroxamate assayare the reason for the lack of reduction of activity of PEGylated enzymein this assay. All of these explanations are provided without wishing tobe limited by a single hypothesis.

These results support the disparate effects of PEGylation upon formationof a hydrogel than on the enzyme's activity in solution. Without wishingto be limited by a single hypothesis, it is believed that thesedifferent effects occur as a result of the increased apparent sizeand/or perceived volume (other than caused by increased size) of theenzyme, which in turn provide beneficial effects for controllingformation of a hydrogel. In any case, according to at least someembodiments of the present invention, the differential effect ofPEGylation enables crosslinking to be controlled during formation of ahydrogel.

EXAMPLE 6 Elution Profile of PEGylated and Non-PEGylated mTG from Gelsof Gelatin

Materials:

-   Gelatin (Pig skin type A, 275 bloom) was from Gelita-   30% Acrylamide/Bis 29:1 and Bio-Safe Coomassie G-250 stain were from    Bio-Rad.-   Molecular weight marker was Precision Plus Dual Color (Bio-Rad)

A crosslinked gelatin gel was made by mixing 0.67 ml of an enzyme mixcomprising of 1:1 mixture of PEGylated mTG (The reaction conditions weresimilar to those in lane 6 in FIGS. 1) and 20 u/ml mTG with 1.33 ml ofgelatin solution (25% gelatin, 3.8 M urea, 0.15 M CaCl₂, 0.1M Na acetatepH 6). The resulting gel was wrapped in saran wrap and incubated at 37°C. for 2 hours. Next, the gel was placed in a tube containing 10 mlsaline and was incubated for 4 hours at 37° C. shaker incubator. Sampleswere taken every hour. Samples were concentrated using Amicon Ultra-4Centrifugal Filter Unit MWCO 30,000 (Millipore), denatured by heating at90° C. in the presence of SDS and beta mercaptoethanol and were analyzedusing SDS-PAGE (8% resolving gel, 4% stacking gel, Mini-Proteanelectrophoresis system, BioRad). To visualize the proteins the gel wasstained with Bio-Safe Coomassie G-250 stain followed by destaining withwater.

In order to quantitate the intensities of the bands in SDS-PAGE, the gelwas scanned with CanoScan 8800F scanner and the resulting image, shownin FIG. 4, was analyzed using Quantity One software (Bio-Rad). The laneassignments are as follows: Lane 1: 10 μl sample taken at t=1 hour

-   Lane 2: 20 μl sample taken at t=1 hour-   Lane 3: 10 μl sample taken at t=4 hour-   Lane 4: 20 μl sample taken at t=4 hour-   Lane 5: Molecular size marker-   Lane 6: 7 μl mTG+PEGylated mTG mix-   Lane 7: 3 μl mTG+PEGylated mTG mix-   Lane 8: 1.5 μl mTG+PEGylated mTG mixture

FIG. 4 shows elution of mTG and PEGylated mTG from the same crosslinkedgelatin gel. Table 3 shows the relative amounts of transglutaminaseeluted from the gel.

TABLE 3 % of mTG from total* 1 hr elution 21.6 4 hr elution 37.3 Enzymemix 14.9 *(Total = mTG + PEGylated mTG)

EXAMPLE 7 Elution of Different Types of PEGylated mTG from CrosslinkedGelatin Gels:

Large Scale PEGylation of mTG with Different Concentrations of 2 kD or 5kD PEG-NHS Activated PEG:

-   Reagents:-   mPEG-glutaryl-NHS, MW 5000 (SunBright ME-050GS, NOF corporation,    Japan)-   mPEG-succinyl-NHS, MW 2000 (SunBright ME-020CS, NOF corporation,    Japan)-   mTG: Ajinimoto active 10% further purified using SP-sepharose ion    exchange chromatography.-   30% Acrylamide/Bis 29:1 and Bio-Safe Coomassie G-250 stain were from    Bio-Rad SDS and beta mercaptoethanol were from Sigma Aldrich

The PEGylation reaction (32 ml) contained 15 u/ml mTG, 100mM HEPES (pH8) and various concentrations of PEG-NHS (2 kD or 5 kD). The reactionswere incubated at room temperature for 2.5 hours, followed by additionof 2.2 ml 1.5 M glycine (97 mM final concentration) in order toneutralize the non-reacted activated PEG. After 15 minutes of furtherincubation at room temperature the reaction mix was concentrated down to8 ml using Vivaspin 20 (Sartorius) while at the same time the reactionbuffer was changed to 0.2 M Na citrate pH 6.

1 volume of different PEGylated mTGs (described above) were mixed with 2volumes of gelatin formulation (25% gelatin, 3.8 M urea, 0.15 M CaCl₂,0.1 M Na acetate pH 6). The mixtures were poured into Teflon coated dogbone shaped molds. After gelation occurred, the gels were taken out ofthe molds, weighed, placed in a closed test tube to prevent drying andincubated at 37° C. for 3 hours. Next, exactly 5 ml of Na citrate pH 6were added for each gram of gel, and the test tube was incubated at a37° C. air shaker at 100 rpm. 0.5 ml samples were taken after 1 hr, 2 hr3 hr and after further incubation for 18 hr at 30° C. .

Samples from each timepoint were denatured by heating at 90° C. in thepresence of SDS and beta mercaptoethanol and were analyzed usingSDS-PAGE (8% resolving gel, 4% stacking gel, Mini-Proteanelectrophoresis system, BioRad). To visualize the proteins the gel wasstained with Bio-Safe Coomassie G-250 stain followed by destaining withwater. In order to quantitate the intensities of the bands in SDS-PAGE,the gel was scanned with CanoScan 8800F scanner and the resulting image,shown in FIG. 5, was analyzed using Quantity One software (Bio-Rad).

The maximal theoretical amount of enzyme that would have been releasedwas loaded on the SDS-PAGE as well and was taken as 100% release. Theactual elution samples were ran side by side and the intensities of thebands were calculated relative to the 100% release. In order toquantitate the intensities of the bands in SDS-PAGE, the gel was scannedwith CanoScan 8800F scanner and the resulting image was analyzed usingQuantity One software (Bio-Rad).

FIG. 5 shows elution of mTG (left) and PEGylated mTG (right) fromdifferent crosslinked gelatin gels. The lane assignments are givenbelow.

-   Lane 1: mTG, 100% release reference-   Lane 2: mTG released from crosslinked gelatin gel, 1 hr time-point-   Lane 3: mTG released from crosslinked gelatin gel, 2 hr time-point-   Lane 4: mTG released from crosslinked gelatin gel, 3 hr time-point-   Lane 5: mTG released from crosslinked gelatin gel, 18 hr time-point-   Lane 6: PEGylated mTG (7 mg/ml PEG-NHS, 5 kD)-100% release reference-   Lane 7: PEGylated mTG (7 mg/ml PEG-NHS, 5 kD) released from gelatin    gel, 1 hr time-point-   Lane 8: PEGylated mTG (7 mg/ml PEG-NHS, 5 kD) released from gelatin    gel, 2 hr time-point-   Lane 9: PEGylated mTG (7 mg/ml PEG-NHS, 5 kD) released from gelatin    gel, 3 hr time-point-   Lane 10: PEGylated mTG (7 mg/ml PEG-NHS, 5 kD) released from gelatin    gel, 18 hr time-point

TABLE 4 % elution from gelatin gels of different types of PEGylated mTG.Total release amounts are shown in Table 4 below. % Elution from gelafter 18 hr Non-PEGylated mTG 26.7 PEGylated mTG (7 mg/ml PEG 5 kD) 12.7PEGylated mTG (14 mg/ml PEG 5 kD) 16.1 PEGylated mTG (7 mg/ml PEG 2 kD)31.5 PEGylated mTG (14 mg/ml PEG 2 kD) 30.9

EXAMPLE 8 Activity of mTG Eluted from Crosslinked Gels

9 ml of Non-PEGylated mTG which was eluted from gelatin gels for 18hours (see Example 7) was concentrated to 0.47 ml using Vivaspin 20(MWCO 30,000; Sartorious). The activity of the concentrated enzyme wasdetermined using the hydroxamate assay as described in Example 5.

The measured activity was found to be 3.65 u/ml.

The calculated activity (based on initial activity in the gel of 5 u/mland % release at 18 hr according to SDS-PAGE in FIG. 5 and itsquantitation in Table 4 of 26.7%) is 4.24 u/ml.

EXAMPLE 9 Mechanical Testing of Gelatin Gels Crosslinked with PEGylatedor Non-PEGylated mTG.

-   Urea, Na citrate, Na Acetate and calcium chloride were from Sigma    Aldrich.-   Gelatin (Pig skin Type A 275 bloom) was from Gelita.-   mTG was from Ajinimoto activa 10% further purified using    SP-sepharose ion exchange chromatography. Activity: 604 units/ml in    0.2M sodium citrate pH 6.-   PEGylated mTG (either 2 kD or 5 kD PEG-NHS) with various degrees of    PEGylation was prepared as described in Example 7.

1 part of PEGylated mTG solution was mixed with 2 parts of gelatinsolution (25% gelatin, 3.8 M urea, 0.15 M CaCl₂, 0.1 M Na acetate pH 6).The mixture was poured into a Teflon-coated dog bone shaped mold. Aftergelation occurred, the gels were taken out of the molds, submerged insaline and incubated at 37° C. for 4 hours. The dimensions of thedogbone-shaped gel were then measured using a digital caliper. Controlsamples were made using 1 part of 15 u/ml of non-PEGylated mTG and 2parts of gelatin solution. For both types of samples, the followingtesting protocol was followed:

The sample was clamped into a tensile testing system (Instron model3343) such that the gel sample between the clamps was approximately 12(width)×1.9 (thickness)×20 (length) mm. The precise dimensions of eachsample were measured immediately prior to tensile testing and thesemeasured values were used to calculate the material properties of thesamples. Following clamping and measuring, tension was applied to eachsample at a rate of 0.25 mm/s until a pre-load of 0.025 N was achieved.This was considered the 0% strain point. Following the preload, tensilestrain was continuously applied to the sample at a rate of 0.5 mm/suntil the sample failed by fracture.

The maximum strain and stress occurred at the fracture point such thatthe ultimate tensile strain and ultimate tensile stress were recorded atthe point of fracture as Strain to break (%) and stress to break (kPa).Elastic modulus was calculated from the linear region between 10% and30% strain for each sample.

Each type of crosslinked gelatin gel was tested with 5 repetitions andthe average and standard deviations are summarized in Table 5.

TABLE 5 Gelation Young's time Modulus Tensile stress at Tensile strain(min) (kPA) break (kPa) at break (%) Control 4.5 92.4 ± 7.4 42.5 ± 2.9 47.8 ± 4.9  (15 u/ml)  7-2 4.5   72 ± 2.9   52 ± 13.6 81.9 ± 24.7 14-26.5   46 ± 4.6 36.6 ± 6.5  88.9 ± 19.8  7-5 3.5 90.4 ± 2.8 76.9 ± 11.698.9 ± 16.3 14-5 4.25 64.2 ± 5.6 77.7 ± 25.5 159.9 ± 52.6  28-5 5.5 47.6± 2.8 79.3 ± 16.5 221.7 ± 59.6 

Table 5 shows the mechanical testing of various types of PEGylated mTG.The left most column refers to the conditions of PEGylation, given asA-B; the value of A as 7 refers to 7 mg/ml PEG, the value of A as 14refers to 14 mg/ml PEG, and the value of A as 28 refers to 28 mg/ml PEG;the value of B as 2 refers to 2 kD PEG, while the value of

B as 5 refers to 5 kD PEG. As shown, increased amounts of PEG result inincreased gelation time and reduced Young's modulus; however, increasedPEG size results in increased tensile strength and increased flexibilityof the resultant gel.

EXAMPLE 10 Performance of Sealant on Living Tissue Using the BurstPressure Test

Porcine small intestine tissues were cleaned of residual material andcut into 10 cm pieces. In each piece a 14 gauge needle puncture wasmade. The tissues were then be soaked in a saline solution and incubatedat 37° C. Prior to applying the sealant material, which was prepared asdescribed in Example 7, the tissue was flattened and the applicationsite of each tissue was blotted using a gauze pad. Approximately 0.1-0.2mL of tested sealant was applied on each application site using a 1 mLsyringe. Within 5 min of the application the tissue was washed withsaline and incubated at 37° C., for 4 hours. Each test group wasexamined in triplicates or more.

For the burst pressure test, the tissue were placed in the Perspex Box,one side tightly sealed (using a clamp) and the other connected to thepressure meter and hand pump (using a plastic restraint). The Perspexbox was filled with saline so that tissue sample is totally submerged.Air was pumped, using the hand-pump at a constant rate (20 mL/min).Burst pressure was determined by the appearance of bubbles.

The results are shown in FIG. 6, indicating burst pressure values forgelatin sealant made with non-PEGylated mTG and 2 types of PEGylatedmTG. As shown, the median results indicate an increase in burst pressurestrength for both types of PEGylated mTG, although a somewhat greatereffect is shown for the more moderately PEGylated enzyme.

EXAMPLE 11 Use of Sealant for Staple Line Reinforcement for in vivoPorcine Model

A Covidien EEA circular surgical stapler was used to perform a circularanastomosis in the rectum of a pig.

Surgical sealant comprised of gelatin solution and PEGylated TG wasprepared as in example 7, with 28 mg/ml 5 kDa PEG-NHS in a reactionvolume of 72 ml. The reaction mix was concentrated using Viva-Spin 20MWCO 30,000 (Sartorius) to 3 ml, such that the activity of theconcentrated PEGylated enzyme was equivalent to 40 u/ml of non-PEGylatedenzyme. 4 mL of sealant (comprised of 2.66 ml gelatin solution and 1.33PEGylated enzyme solution) was applied evenly around the circumferenceof the rectal staple-line and left to cure for 4 minutes. The animal wasthen closed.

14 days post-surgery, the pig was sacrificed. The sealed anastomoticarea was examined for gross pathology and the sealant was palpated toqualitatively assess its mechanical properties.

Result:

The sealant did not undergo significant degradation over the course ofthe 14 day implantation period. It remained strongly adhered to thestaple line, maintaining 100% integrity over the length of the stapleline. The sealant material was pliable and flexible, matching the shapeand movement of the circular staple-line shape. No inflammation orabdominal adhesions were noted in the area of the sealant or stapleline. The anastomosis was fully healed with no signs of leakage. Nostrictures were observed in the rectum.

EXAMPLE 12 Non-covalent Binding of Cross-linking Enzyme to InsolubleCarrier

SP sepharose was bound to mTG (microbial transglutaminase) and a gel wasmade. Gelation occurred in 16-23 minutes with immobilizedtransglutaminase, while soluble enzyme caused gelation to occur in lessthan 6 minutes. Immobilization therefore increased the time required forgelation.

500 μl washed SP sepharose beads (GE Healthcare) were mixed with 2.7 ml13.5 mg/ml of purified mTG with 11.55 ml 50 mM Na AC pH 5.5 (15 mltotal).

The mixture was incubated in a shaker at room temperature for 20minutes. The beads were then washed 3 times with 11.5 ml 50mM NaAc pH5.5, 3 minutes each wash. 70% of the protein was bound to the beadsafter the washing step. The beads were resuspended in 9.5ml 50mM NaAc pH5.5 to a final volume of 10 ml. The mTG-loaded beads were mixed with50mM NaAc pH 5.5 in various compositions in a final volume of 600 μl asfollows (in parenthesis the amount of bound mTG and the calculatedtheoretical mTG activity based on the measured activity of 1 mg unboundmTG - 33 hydroxamate units):

A: 292 μl beads+308 NaAc (1.244 mg/ml=41 u/ml)

B: 400 μl beads+200 μl NaAc (1.704 mg/ml=56.2 u/ml))

C: 500μl beads+100 μl NaAc (2.13 mg/ml=70.3 u/ml)

D: 550 μl beads+50 μl NaAC (2.34 mg/ml=77.2 u/ml)

500 μl from each reaction A-D were mixed with 1 ml of 25% gelatinsolution in sodium acetate buffer with 4.5 M urea,) with syringe tosyringe mixing. Gelation time was determined as the time in which thegelatin ceased to flow by visual inspection.

Gelation time:

A: about 23 min

B: about 21 min

C: about 20 min

D: about 16 min

Control (unbound mTG 10 u/ml): 5.5 min

Gelation times with bound mTG were significantly slower compared to freeenzyme. This suggests that binding of enzyme to a larger scaffold orinsoluble carrier slows the mobility of the enzyme in a hydrogel matrixwith the result that gelation, a sign of increased mechanical stiffness,is achieved at a later time point through cross-linking by bound enzymeas compared to free (unbound) enzyme. Thus, the enzyme binding resultedin modified mechanical properties of the hydrogel matrix.

EXAMPLE 13 Enzyme Modification with Oxidized Dextran

This experiment demonstrates that modification of enzyme by bindinglarge soluble molecule can result in modification of mechanicalproperties.

Methods

1 gram dextran was dissolved in 20 ml purified water. 1.3 gram sodiumperiodate was added and the reaction stirred at room temperatureprotected from light by aluminum foil for 80 minutes (9:50-11:10).

2 gram glycerol were added to quench the non-reacted periodate.

The reaction was dialyzed 3 times against 1 L PuW for 2:00 hr, withwater change in between.

TABLE 6 Conjugation_of mTG to oxidized dextran: 1M NaBH₄CN mTG:dextranOxidized phosphate Distilled 250 mg/ml in ratio mTG dextran pH 6.0 waterPuW A 1:4 0.75 ml 0.8 ml 0.4 1.05 0.1 ml (25 mg) (10 mg) (36.4 mg) B 1:10.75 ml 0.2 ml 0.4 1.65 0.1 ml (25 mg) (10 mg) (9.1 mg) C 4:1 0.75 ml0.05 ml 0.4 1.8 0.1 ml (25 mg) (10 mg) (2.3 mg) D 10:1  0.75 ml 0.02 ml0.4 1.83 0.1 ml (25 mg) (10 mg) (0.91 mg)

The reactions were incubated at room temperature overnight and then werepurified by diafiltration using vivaspin 20 (Sartorius).

Results

FIG. 7 shows SDS-PAGE analysis of conjugation reactions A-D. Thefollowing amounts of dextran-conjugated mTG were loaded on a 4-15%Mini-Protean TGX gel (Bio-Rad): 4.35 μg (Reaction A), 4.38 μg (ReactionB), 1.98 μg (Reaction C) and 3 μg (Reaction D). The samples contained0.1% SDS but no reducing agent and were heated at 85° C. for 10 minutesbefore loading. The gel was run at a constant voltage (200V) and theprotein bands were visualized by staining with Bio-Safe Coomassie G-250solution (Bio-Rad). The molecular weight marker was Precision Plus(Bio-Rad). The example shows that it is possible to immobilize acrosslinking enzyme, in this case mTG (microbial transglutaminase), on asoluble polymer. Furthermore, at higher dextran:mTG ratios, moremolecules of free mTG are converted to high MW conjugates with dextran.

EXAMPLE 14 Horseradish Peroxidase PEGylation

This experiment demonstrates that modification of Horseradish Peroxidase(another crosslinking enzyme) by PEGylation can modify matrices formedby peroxidase crosslinking. In another embodiment of the presentinvention, the crosslinking enzyme is horseradish peroxidase (HRP) andHRP is modified by attachment of PEG molecules to the HRP molecules inorder to modify the mechanical properties of the gelatin hydrogel formedby HRP crosslinking.

Methods

-   Preparation of phenol-modified gelatin (gelatin-Ph): Two grams of    high molecular weight gelatin Type A were dissolved in 100 ml 50 mM    MES (2-(N-morpholino)ethanesulfonic acid; Sigma Aldrich) buffer    pH 6. To this 2% w/w solution the following reagents were added:    0.984 gram tyramine (Sigma Aldrich). 0.218 gram NHS    (N-Hydroxysuccinimide; Sigma Aldrich), 0.72 gram EDC    (1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide; Sigma Aldrich). The    reaction was stirred at room temperature for 16 hours, and then    dialyzed extensively against distilled water. The dialyzate was    freeze dried and the resulting dry foam was dissolved in 0.1 M    phosphate buffer pH 6.0 to a final volume of 16 ml or 12.5% w/w    gelatin.-   PEGylation of HRP: 2 mg/ml HRP Type I (Sigma, St Louis, Mo.) were    reacted with 60 mg/ml PEG-NHS 5 kD in 100 mM Hepes pH 8.0 for 2    hours, followed by addition of 110 mM glycine to quench the    non-reacted PEG-NHS and 30 minutes further incubation. The PEGylated    HRP was purified by extensive dialysis against 25 mM phosphate    buffer pH 6.0.-   HRP and PEGylated HRP dependent gelation of gelatin-Ph: Gelatin    component: 5 ml gelatin-Ph+0.5 ml 20 mM H₂O₂ mixed in a glass vial :    4.4 ml were transferred to syringe A. HRP/PEGylated HRP component: 1    ml 0.035 mg/ml HRPor PEGylated HRP in Syringe B. The gelatin and    enzyme components were mixed by syringe to syringe transfer and then    incubated at 37° C. while being inverted to determine gelation time.

After 20 minutes the gels were weighed, covered with 10 ml saline andincubated at 37° C. for 16 hours, after which the gels were weighedagain to determine swelling ratio.

Results

After mixing, the gelatin and enzyme mixture formed a gel within 3minutes. SDS-PAGE analysis for HRP and PEGylated HRP proteins can beseen in FIG. 8. HRP and PEGylated HRP (20 μg of each) were loaded on a4-15% Mini-Protean TGX gel (Bio-Rad). The samples contained 0.1% SDS butno reducing agent and were heated at 85° C. for 10 minutes beforeloading. The gel was run at a constant voltage (200V) and the proteinbands were visualized by staining with Bio-Safe Coomassie G-250 solution(Bio-Rad). The molecular weight marker was Precision Plus (Bio-Rad)

The measured swelling ratios are detailed below in Table 7:

TABLE 7 Weight 20 minutes Weight after over- % swelling after gelation(A) night at 37deg C. (B) (B − A)/A × 100 HRP gel 1 1.82 3.01 65.4 HRPgel 2 1.33 2.29 72.2 HRP gel 3 1.59 2.58 62.3 PEGylated 1.69 3.62 114.2HRP gel 1 PEGylated 1.73 3.68 112.7 HRP gel 2

As can be seen in Table 7 above, the gels made with PEGylated HRPswelled to a larger extent than gels made with non-PEGylated HRP. Thisdemonstrates that the mechanical properties of the gelatin hydrogelsformed by the PEGylated (modified) HRP were significantly different thanthe mechanical properties of the hydrogels formed by the free(unmodified) HRP. Both types of gels were heat resistant and did notdissolve after 1 hour at 80 deg C.

EXAMPLE 15 Effect of Partial PEGylation

Cross-linking enzyme with pegylation to different degrees resulted indifferent degrees of mechanical properties. This example demonstrateshow the mechanical properties of a enzymatically crosslinked hydrogelcan be specifically controlled by modulating the hydrodynamic volume, inthis case the degree of PEGylation, such that greater hydrodynamicvolume (i.e. more PEGylation) results in a more elastic matrix and lesshydrodynamic volume (i.e. less PEGylation results in a less elasticmatrix. Naturally, the unmodified hydrodynamic volume (i.e. noPEGylation) results in the least elastic matrix. Instron data andSDS-Page gel data are described below with regard to these effects.

Methods

Three PEGylation reactions were performed side by side. The reactionswere done at room temperature for 2.5 hr in 100 mM HEPES pH 8.0 usingPEG-NHS 5K . Following the reaction, the unreacted excess PEG wasneutralized with 110 mM glycine and incubation continued for 30 moreminutes.

Reaction A and B had the same PEG:amine ratio but in A, both the mTG andthe PEG were 3× more concentrated than in B. Reaction C is similar to Abut the PEG:amine ratio was half the ratio in A. The results are shownin Table 8.

TABLE 8 A B C PEG conc (mg/ml) 21.00 7.00 10.50 PEG conc (mM) 4.20 1.402.10 mTG conc (mg/ml) 5.96 2.00 5.95 mTG amine conc (mM) 3.15 1.05 3.14ratio PEG/amine 1.33 1.33 0.67

Following the completion of these reactions, each resulting solution ofPEGylated mTG solution was reacted with a 25% gelatin solution (insodium acetate buffer with 4.5 M urea) at a 1:2 ratio, mTG solution togelatin solution, to form a gelatin hydrogel. The mTG activity levels ofeach PEGylated mTG solution were normalized such that the reaction timewith the gelatin was identical for all groups. Following the formationof each hydrogel, it was cultured at 37° C. for 2 hours and thenmechanically tested using a tensile testing system.

Results are shown in FIG. 9, which is an image of SDS-PAGE analysis ofPEGylated mTG. PEGylated mTG from reactions A, B and C (5 μg of each)were loaded on a 6% polyacrylamide gel and subjected to SDS-PAGE. Thesamples contained 0.1% SDS but no reducing agent and were heated at 85°C. for 10 minutes before loading. The gel was run at a constant voltage(200V) and the protein bands were visualized by staining with Bio-SafeCoomassie G-250 solution (Bio-Rad). The molecular weight marker wasPrecision Plus (Bio-Rad).

The SDS-PAGE profile shows how reactions A, B, and C resulted in mTGmolecules bound with PEG molecules to different degrees such that manyPEG molecules are bound to the mTG in A, fewer in B, and even fewer inC.

The varying degrees of PEGylation significantly affect the mechanicalproperties of the gelatin matrices as can be seen by the below resultswherein the most PEGylated mTG, A, results in the most elastic (highesttensile strain at break) hydrogel and the non-PEGylated mTG results inthe least elastic hydrogel, with the partially PEGylated mTG groups, Band C, falling out in between in correlation to each groups respectivedegree of PEGylation.

TABLE 9 PEGylated PEGylated PEGylated Control mTG mTG mTG (non- formulaA formula B formula C PEGylated) Modulus 89.14 118.97 135.536 153.27(kPa) Tensile 123.86 122.96 105.629 83.976 stress at break (kPa) Tensile193.11 125.11 87.571 63.863 strain at break (%)

EXAMPLE 16 Free PEG has no Effect on Gelation

As a control, Instron results of mechanical properties of gels weretested with and without free PEG. By “free” it is meant that the PEGmolecule was placed in solution with the crosslinking enzyme, but wasnot covalently bound to the enzyme. The results showed that free PEGdoes not result in the mechanical property modifications brought aboutby covalent binding of PEG to the cross-linking enzyme (i.e.modification of the enzyme itself with PEG).

Methods

4 ml aliquots of gelatin solution (25% gelatin, 4.5 M urea in sodiumacetate buffer) with or without 20% PEG 6000 were each mixed with 2 mlaliquots of 15 u/ml mTG. 2 ml of each resulting solution was poured intodog-bone mold as described for Example 9. The resulting gel was takenout of the mold and incubated in saline at 37° C. for 2 hours, followedby tensile testing as described for Example 9.

Results

TABLE 10 +20% −PEG PEG 6000 Modulus 73.72 55.03 (kPa) Tensile 41.7430.13 stress at break (kPa) Tensile 57.7 57 strain at break (%)

The results of Table 10 demonstrate that the addition of free PEG, aplasticizer, had a minimal or no effect on enzyme crosslinked hydrogelmatrix mechanical properties but that these mechanical propertymodifications are minor in comparison with the modifications achieved byincreasing the hydrodynamic volume of the enzyme molecules through theattachment of PEG to the enzyme. In particular, the elasticity (strainto break) of the matrix was not improved at all by addition of free PEG,whereas PEGylation of the enzyme molecules results in a significantincrease in matrix elasticity, as can be seen in several other examples.

EXAMPLE 17 Mixed Modified/Non-modified Cross-linking Enzyme

Various mixtures of modified enzyme mixed with non-modified enzyme weretested. Different levels of modification of various mechanicalproperties can be obtained according to the specific mixture.

In another embodiment of the present invention, modified enzyme is usedtogether with unmodified (free) enzyme in order to achieve mechanicalmodification of an enzyme-crosslinked matrix.

Methods

4 ml gelatin solution aliquots (25% gelatin, 4.5 M urea, sodium acetatebuffer) with or without 20% PEG 6000 were mixed with 2 ml 55 u/mlPEGylated mTG with or without additional non-PEGylated mTG and 2 ml ofeach resulting solution was poured into a dog-bone mold as described forExample 9. The resulting gel was taken out of the mold and incubated insaline at 37° C. for 24 hours, followed by tensile testing as describedfor Example 9.

Results

TABLE 11 55 u/ml 55 u/ml 55 u/ml PEGylated PEGylated PEGylated mTG + 5u/ml mTG + 10 u/ml mTG free mTG free mTG Modulus 73.5 134.88 163.06(kPa) Tensile 101.17 89.64 87.66 stress at break (kPa) Tensile 170.5174.73 57.65 strain at break (%)

The results of Table 11 indicate that mechanical properties of an enzymecrosslinked hydrogel can be modified both by using only modified enzymeand also, to a lesser degree, by using a mixture of modified enzyme withfree enzyme.

EXAMPLE 18 Bi-functional PEG-enzyme Bridges

This experiment demonstrated cross-linking of enzyme to itself through abi-functional PEG bridge. For this example, two or more enzyme moleculescan be bound to each other to increase the overall hydrodynamic volumeof the enzyme aggregate. One way of accomplishing this is by using abi-functional molecule that forms a bridge between enzyme molecules.

Methods

mTG (15 u/ml, 0.5 mg/ml) was incubated with various concentrations of 10kD bifunctional PEG-NHS in 100 mM Hepes pH8 at room temperature for 2hours, followed by addition of 110 mM glycine for 30 more minutes toneutralize the excess non-reacted PEG. The specific conditions are shownin Table 12 below.

TABLE 12 PEG concentration Reaction # (mg/ml) PEG:amine ratio A 0.780.296 B 1.56 0.59 C 3.125 1.18 D 6.25 2.36

Following the reaction, 5 μg of enzyme each reaction composition wasloaded on a 7.5% polyacrylamide gel and subjected to SDS-PAGE analysis.The samples contained 0.1% SDS but no reducing agent and were heated at85° C. for 10 minutes before loading. The gel was run at a constantvoltage (200V) and the protein bands were visualized by staining withBio-Safe Coomassie G-250 solution (Bio-Rad). The molecular weight markerwas Precision Plus (Bio-Rad).

For mechanical property testing, 4 ml aliquots of gelatin solution (25%gelatin, 4.5 M urea, sodium acetate buffer) were mixed with 2 mlaliquots of 15 u/ml either free mTG or PEGylated mTG (reaction C). 2 mlof the resulting solution were poured into dog-bone molds as describedfor Example 9. The resulting gel was taken out of the mold and incubatedin saline at 37° C. for 24 hours, followed by tensile testing asdescribed for Example 9.

Results

The SDS-PAGE results of FIG. 10 shows that at relatively low PEG:mTGratios, some of the mTG was converted to very high MW products, largerthan PEGylated products obtained in reactions containing similarconcentrations of monofunctional 5 kD PEG. This demonstrates that thehigh MW products consist of multimers of enzyme molecules crosslinked toeach other by a bifunctional PEG bridge and demonstrate the efficacy ofusing bifunctional PEG to modify crosslinker enzyme molecules by bindingthem to each other.

The mechanical testing results below show that the binding of enzymecrosslinking molecules to each other results in significant modificationto the gelatin hydrogels formed by crosslinking with these linked enzymemolecules, as compared with gelatin hydrogels formed by crosslinkingwith free enzyme molecules. The results are shown in Table 13.

TABLE 13 15 u/ml mTG + bifunctional PEG- 15 u/ml mTG 10K Modulus 151.3935.125 (kPa) Tensile 85.08 38.04 stress at break (kPa) Tensile 67.82121.4 strain at break (%)

EXAMPLE 19 Mass Spectrometry Analysis of PEGylated mTG

Three different batches of PEGylated mTG (microbial transglutaminaseenzyme modified with PEG-NHS-5 kD) were analyzed by MALDI-TOF massspectrometry. FIG. 11 shows the m/z spectrum of one of these batches.

Mass Spectrometry

Intact molecular mass measurement was performed on a Bruker Reflex IIImatrix-assisted laser desorption /ionization (MALDI) time-of-flight(TOF) mass spectrometer (Bruker, Bremen, Germany) equipped with delayedion extraction, reflector and a 337 nm nitrogen laser. Each massspectrum was generated from accumulated data of 200 laser shots.External calibration for proteins was achieved by using BSA andmyoglobin proteins (Sigma, St Louis, Mo.).

Sample Preparation for MALDI-TOF MS—Dry Ddroplet Method.

2,5-Dihydroxybenzoic acid (DHB) 0.5 l of volume of matrix in 2:1 0.1%TriFluoroAcetic acid (TFA)—acetonitrile (ACN) and 0.5 l of samplesolution in Formic acid/Isopropanol/H20 (1:3:2) were mixed on the targetand allowed to air dry. After solvent evaporation the samples thesamples were rewashed 1-3 times with 0.1% TFA.

TABLE 14 Average size Calculated PEGylation Average size secondarynumber of batch # main peak peak PEGs 1 82343.63 42202.23 9.16 287430.04 44676.7 11.2 3 84543.16 44937.4 8.97

The results of Table 14 indicate that PEGylation of mTG crosslinkingenzyme with 5 kDa PEG-NHS reagent results in the binding of multiple PEGmolecules to each enzyme molecule.

EXAMPLE 20 PEGylation of mTG at a Fixed PEG to Amine Ratio with VariousConcentrations of Reactants.

This example demonstrates the large effect of total reactantconcentration on the extent of PEGylation. When the ratio of PEG:aminewas maintained at a fixed value, a correlation between the concentrationof reactants (PEG and mTG) and the extent of PEGylation wasdemonstrated.

Methods

PEGylation of mTG with PEG-NHS-5 kD was carried out at room temperaturein 100 mM Hepes pH 8.0 for 2.5 hours, followed by addition of 110 mMglycine to neutralize unreacted PEG-NHS. Following the reaction, 5 μg ofenzyme each reaction composition was loaded on a 6.0% polyacrylamide geland subjected to SDS-PAGE analysis. The samples contained 0.1% SDS butno reducing agent and were heated at 85° C. for 10 minutes beforeloading. The gel was run at a constant voltage (200V) and the proteinbands were visualized by staining with Bio-Safe Coomassie G-250 solution(Bio-Rad). The molecular weight marker was Precision Plus (Bio-Rad).

Reactions were performed according to the below conditions (Table 15):

TABLE 15 A B C D E PEG conc. (mg/ml) 1.75 3.50 7.00 14.00 21.00 PEGconc. (mM) 0.35 0.70 1.40 2.80 4.20 mTG conc. (mg/ml) 0.50 0.99 1.983.97 5.95 mTG amine conc. (mM) 0.26 0.52 1.05 2.09 3.14 ratio PEG/amine1.34 1.34 1.34 1.34 1.34

Results

The ensuing PEGylated mTG molecules can be seen in the SDS-PAGE lanesshown in FIG. 12. As can be seen, even when PEG:Amine ratio was keptfixed, the higher concentration of reactants resulted in product thatwas more PEGylated.

Although selected embodiments of the present invention have been shownand described individually, it is to be understood that any suitableaspects of the described embodiments may be combined, or indeed aplurality of embodiments may be combined.

In addition, although selected embodiments of the present invention havebeen shown and described, it is to be understood the present inventionis not limited to the described embodiments. Instead, it is to beappreciated that changes may be made to these embodiments withoutdeparting from the principles and spirit of the invention, the scope ofwhich is defined by the claims and the equivalents thereof.

What is claimed is:
 1. A cross-linked matrix, comprising a substratepolymer and a modified enzyme molecule, wherein said substrate polymerhas been crosslinked by said modified enzyme molecule, wherein both saidmodified enzyme molecule and said substrate polymer are present in thematrix, said modified enzyme molecule having a modification selectedfrom the group consisting of: a modification that increases the actualsize of said enzyme molecule; or a modification that increases thehydrodynamic volume of said enzyme molecule wherein said modified enzymehas a reduced cross-linking rate in comparison to non-modified enzymethat results in a lesser extent of crosslinking of said substratepolymer in comparison to a matrix formed with non-modified enzyme. 2.The matrix of claim 1, wherein said modification is of the ε-amino groupof lysines of the modified enzyme molecule through a process selectedfrom the group consisting of succinylation (with succinic anhydride),acetylation (with acetic anhydride), carbamylation (with cyanate),reductive alkylation (aldehydes) and treatment with maleic anhydride. 3.The matrix of claim 1, wherein said modification is of one or more sidechains containing carboxylic acids of the modified enzyme molecule todecrease the number of negative charges.
 4. The matrix of claim 1,wherein said modification comprises covalent attachment of a modifyingmolecule to said modified enzyme molecule.
 5. The matrix of claim 4,wherein said modified enzyme molecule has a reduced diffusion rate and areduced cross-linking rate in comparison to non-modified enzyme, whereinsaid reduced cross-linking rate is at least 10% of the non-modifiedenzyme cross-linking rate.
 6. The matrix of claim 5, wherein saidmodifying molecule comprises a carrier or polymer.
 7. The matrix ofclaim 6, wherein said polymer comprises a synthetic polymer, acellulosic polymer, a protein or a polysaccharide.
 8. The matrix ofclaim 7, wherein said cellulosic polymer comprises one or more ofcarboxymethyl cellulose, hydroxypropyl methylcellulose, hydroxyethylcellulose, or methyl cellulose.
 9. The matrix of claim 7, wherein saidpolysaccharide comprises one or more of dextran, chondroitin sulfate,dermatan sulfate, keratan sulfate, heparin, heparan sulfate, hyaluronicacid or a starch derivative.
 10. The matrix of claim 7, wherein saidmodifying molecule comprises PEG (polyethylene glycol).
 11. The matrixof claim 10, further comprising a co-polymer that is not covalentlybound to said modified enzyme molecule or to said substrate polymer. 12.The matrix of claim 11, wherein said co-polymer comprises apolysaccharide or a cellulosic polymer.
 13. The matrix of claim 12,wherein said polysaccharide comprises dextran, chondroitin sulfate,dermatan sulfate, keratan sulfate, heparin, heparan sulfate, hyaluronicacid or a starch derivative.
 14. The matrix of claim 12, wherein saidcellulosic polymer comprises carboxymethyl cellulose, hydroxypropylmethylcellulose, hydroxyethyl cellulose, methyl cellulose.
 15. Thematrix of claim 14, wherein said modified enzyme molecule is modified bycross-linking said modified enzyme molecule to a plurality of othermodified enzyme molecules to form an aggregate of a plurality ofcross-linked enzyme molecules.
 16. The matrix of claim 14, wherein amodification or an extent of modification of said modified enzymemolecule affects at least one property of the matrix selected from thegroup consisting of tensile strength, stiffness, viscosity, elasticity,flexibility, strain to break, stress to break, Poisson's ratio, swellingcapacity and Young's modulus, or a combination thereof.
 17. The matrixof claim 1, wherein an extent of modification of said modified enzymemolecule determines mobility of said modified enzyme molecule in, ordiffusion from, the matrix.
 18. The matrix of claim 17, wherein saidmodification of said modified enzyme molecule reduces diffusioncoefficient of said modified enzyme molecule in a solution of saidmodified enzyme molecule and said substrate polymer or in a matrix ofsaid modified enzyme molecule and said substrate polymer, in comparisonto a solution or matrix of non-modified enzyme molecule and saidsubstrate polymer.
 19. The matrix of claim 1, wherein an extent ofmodification of said modified enzyme molecule determines one or morematrix mechanical properties.
 20. The matrix of claim 1, wherein saidmodified enzyme molecule shows a greater differential of crosslinkingrate in crosslinked polymer than in solution as compared to non-modifiedenzyme molecule.
 21. The matrix of claim 1, wherein said at least onesubstrate polymer comprises a substrate polymer selected from the groupconsisting of a naturally cross-linkable polymer, a partially denaturedpolymer that is cross-linkable by said modified enzyme and a modifiedpolymer comprising a functional group or a peptide that iscross-linkable by said modified enzyme.
 22. The matrix of claim 21,wherein said at least one substrate polymer comprises gelatin, collagen,casein or albumin, or a modified polymer, and wherein said modifiedenzyme molecule comprises a modified transglutaminase and/or a modifiedoxidative enzyme.
 23. The matrix of claim 22, wherein said at least onesubstrate polymer comprises gelatin selected from the group consistingof gelatin obtained by partial hydrolysis of animal tissue or collagenobtained from animal tissue, wherein said animal tissue is selected fromthe group consisting of animal skin, connective tissue, antlers, horns,bones, fish scales, and a recombinant gelatin produced using bacterial,yeast, animal, insect, or plant systems or any type of cell culture, orany combination thereof.
 24. The matrix of claim 23, wherein saidgelatin is of mammalian or fish origin.
 25. The matrix of claim 24,wherein said gelatin is of type A (Acid Treated) or of type B (AlkalineTreated).
 26. The matrix of claim 25, wherein said gelatin is of 250-300bloom.
 27. The matrix of claim 24, wherein said gelatin has an averagemolecular weight of 75-150 kda.
 28. The matrix of claim 22, wherein saidmodified transglutaminase comprises modified microbial transglutaminase.29. The matrix of claim 28, wherein said modified polymer is modified topermit crosslinking by said modified microbial transglutaminase.
 30. Thematrix of claim 22, wherein said modified oxidative enzyme comprises oneor more of tyrosinase, laccase, or peroxidase.
 31. The matrix of claim30, wherein said matrix further comprises a carbohydrate comprising aphenolic acid for being cross-linked by said modified oxidative enzymeas said at least one substrate polymer.
 32. The matrix of claim 31,wherein said carbohydrate comprises one or more of arabinoxylan orpectin.
 33. The matrix of claim 1, wherein said enzyme molecule ismodified through PEGylation and wherein said PEGylation providesimmunogenic masking by masking said enzyme molecule from an immunesystem of a host animal receiving the matrix.
 34. The matrix of claim33, wherein said host animal is human.
 35. A method for sealing a tissueagainst leakage of a body fluid, comprising applying a matrix of claim 1to the tissue.
 36. The method of claim 35, wherein said body fluidcomprises blood, such that said matrix is a hemostatic agent.
 37. Ahemostatic agent or surgical sealant, comprising a matrix of claim 1.38. A composition for sealing a wound, comprising a matrix of claim 1.39. A method of treatment of sealing a tissue in a subject in needthereof, comprising applying the composition of claim 38 to suture orstaple lines in the tissue, for sealing said suture or staple lines insaid tissue.
 40. A composition for a vehicle for localized drugdelivery, comprising a matrix of claim
 1. 41. A composition for tissueengineering, comprising a matrix of claim 1, adapted as an injectablescaffold.
 42. A cross-linked matrix, comprising a substrate polymer anda modified enzyme molecule, wherein said substrate polymer has beencrosslinked by said modified enzyme molecule, wherein both said modifiedenzyme and said substrate polymer are present in the matrix, saidmodified enzyme molecule having a modification that modifies anelectrostatic charge of said modified enzyme molecule to be of oppositesign to a net charge of said substrate polymer by changing theisoelectric point (pI) of said modified enzyme in comparison tounmodified enzyme, wherein said modification reduces the crosslinkingrate in comparison to non-modified enzyme resulting in a lesser extentof crosslinking of said substrate polymer in comparison to a matrixformed with non-modified enzyme.
 43. A cross-linked matrix, comprising asubstrate polymer and a modified enzyme molecule, wherein said substratepolymer has been crosslinked by said modified enzyme molecule, whereinboth said modified enzyme and said substrate polymer are present in thematrix, said modified enzyme molecule having a modification such thatsaid modified enzyme molecule has a reduced cross-linking rate incomparison to non-modified enzyme that results in a lesser extent ofcrosslinking of said substrate polymer in comparison to a matrix formedwith non-modified enzyme, wherein said reduced cross-linking rate is atleast 10% of the non-modified enzyme cross-linking rate.
 44. Across-linked matrix, comprising a substrate polymer and a modifiedenzyme molecule, wherein said substrate polymer has been crosslinked bysaid modified enzyme molecule, wherein both said modified enzyme andsaid substrate polymer are present in the matrix, said modified enzymemolecule being modified by applying an activated PEG to an enzymemolecule and thereby producing a PEGylated enzyme molecule as saidmodified enzyme molecule, wherein said activated PEG comprises methoxyPEG (mPEG), its derivatives, mPEG-NHS, succinimidyl (NHS) esters of mPEG(mPEG-succinate-NHS), mPEG-glutarate-NHS, mPEG-valerate-NHS,mPEG-carbonate-NHS, mPEG-carboxymethyl-NHS, mPEG-propionate-NHS,mPEG-carboxypentyl-NHS), mPEG-nitrophenylcarbonate, mPEG-propylaldehyde,mPEG-Tosylate, mP EG-carbonylimidazole, mPEG-isocyanate or mPEG-epoxide,wherein said modification reduces the crosslinking rate in comparison tonon-modified enzyme resulting in a lesser extent of crosslinking of saidsubstrate polymer in comparison to a matrix formed with non-modifiedenzyme .
 45. The matrix of claim 44, wherein said activated PEG reactswith amine groups or thiol groups on said enzyme molecule.
 46. Thematrix of claim 45, wherein the molar ratio of said activated PEG tolysine residues of said enzyme molecule is in a range of from 0.5 to 25.47. The matrix of claim 46, wherein said activated PEG ismonofunctional, heterobifunctional, homobifunctional, ormultifunctional.
 48. The matrix of claim 47, wherein said activated PEGis branched PEGs or multi-arm PEGs.
 49. The matrix of claim 48, whereinsaid activated PEG has a size ranging from 1000 dalton to 40,000 dalton.50. A method for controlling formation of a matrix, comprising modifyingan enzyme molecule with a modification selected from the groupconsisting of: a modification that increases the actual size of saidenzyme molecule; or a modification that increases the hydrodynamicvolume of said modified enzyme molecule; mixing said modified enzymemolecule with at least one substrate polymer that is a substrate of saidmodified enzyme molecule; and forming the matrix through crosslinking ofsaid at least one substrate polymer by the action of said modifiedenzyme molecule, wherein said modification reduces the crosslinking ratein comparision to non-modified enzyme resulting in a lesser extent ofcrosslinking of said substrate polymer in comparison to a matrix formedwith non-modified enzyme, and wherein said forming the matrix is atleast partially controlled by said modification of said enzyme moleculeand wherein said matrix comprises said modified enzyme molecule andcross-linked substrate polymer.
 51. The method of claim 50, wherein saidmodification reduces a crosslinking rate of said modified enzymemolecule as an extent of crosslinking of said at least one substratepolymer increases.
 52. The method of claim 51, wherein said modifiedenzyme molecule and said at least one substrate polymer are mixed insolution, such that said modification controls extent of crosslinking ofsaid at least one substrate polymer as a viscosity of said solutionincreases.
 53. The method of claim 52, wherein said modifying comprisesPEGylation of the enzyme at a pH in a range from 7 to
 9. 54. The methodof claim 53, wherein pH of the PEGylation reaction is 7.5-8.5.